diff --git a/docs/sub_mask.md b/docs/sub_mask.md
new file mode 100644
index 000000000..ea1e1ad5b
--- /dev/null
+++ b/docs/sub_mask.md
@@ -0,0 +1,33 @@
+## Sub Mask
+
+Sample circular regions from a mask.
+
+**plantcv.plantcv.sub_mask**(*img, mask, num_masks=1, radius=5*)
+
+**returns** A `numpy.ndarray` labelled mask.
+
+- **Parameters:**
+    - img - Grayscale or RGB image.
+	- mask - Binary mask of the image.
+	- num_masks - A number of circular regions to make masks of, defaults to 1. These spots cannot overlap, so specifying too many may trigger a warning that fewer masks could be placed than were specified.
+	- radius - Radius of the circular region(s) to select. These spots cannot overlap, so specifying too large of a radius may trigger a warning that fewer masks could be placed than were specified.
+
+
+- **Context:**
+	- Used to downsample an image, particularly for spectral analysis.
+
+
+- **Example use:**
+	- Below
+
+
+
+```python
+
+from plantcv import plantcv as pcv
+
+spot_masks = pcv.sub_mask(img, mask, num_masks=2, radius=5)
+
+```
+
+**Source Code:** [Here](https://github.com/danforthcenter/plantcv/blob/main/plantcv/plantcv/submask.py)
diff --git a/mkdocs.yml b/mkdocs.yml
index d41c85b24..71ac913e6 100644
--- a/mkdocs.yml
+++ b/mkdocs.yml
@@ -46,6 +46,7 @@ nav:
       - 'Auto Crop': auto_crop.md
       - 'Background Subtraction': background_subtraction.md
       - 'Canny Edge Detection': canny_edge_detect.md
+      - 'Circular Sub Masks': sub_mask.md
       - 'Closing': closing.md
       - 'Color Palette': color_palette.md
       - 'Colorspace Conversion':
diff --git a/plantcv/plantcv/__init__.py b/plantcv/plantcv/__init__.py
index 0741a7343..cb70abbab 100644
--- a/plantcv/plantcv/__init__.py
+++ b/plantcv/plantcv/__init__.py
@@ -85,6 +85,7 @@
 from plantcv.plantcv.process_results import process_results
 from plantcv.plantcv.json2csv import json2csv
 from plantcv.plantcv.masks2labels import masks2labels
+from plantcv.plantcv.submask import sub_mask
 # add new functions to end of lists
 
 __all__ = [
@@ -167,5 +168,6 @@
     "qc",
     "process_results",
     "json2csv",
-    "masks2labels"
+    "masks2labels",
+    "sub_mask"
 ]
diff --git a/plantcv/plantcv/submask.py b/plantcv/plantcv/submask.py
new file mode 100644
index 000000000..6e83e9316
--- /dev/null
+++ b/plantcv/plantcv/submask.py
@@ -0,0 +1,106 @@
+from plantcv.plantcv.roi.roi_methods import circle
+from plantcv.plantcv.roi.roi2mask import roi2mask
+from plantcv.plantcv.warn import warn
+import numpy as np
+import random
+import cv2
+
+
+def sub_mask(img, mask, num_masks=1, radius=5):
+    """
+    Make circular sub-masks inside the mask of an object
+
+    Parameters
+    ----------
+    img       = numpy.ndarray, input image
+    mask      = numpy.ndarray, binary mask of object
+    num_masks = int, number of circular sub-masks to make
+    radius    = int, radius of circular mask to make. Defaults to 5.
+
+    Returns
+    -------
+    labeled_mask = numpy.ndarray, labelled mask of circular masks each within complete mask.
+    """
+    # Create an empty mask
+    labeled_mask = np.zeros_like(mask)
+    tries = 0
+    sample_num = 0
+    while len(np.unique(labeled_mask)) - 1 < num_masks:
+        tries += 1
+        spot = _make_random_circle(img=img, mask=mask, radius=radius)
+        spot_mask = roi2mask(img=img, roi=spot)
+        within = _is_mask_within(full_mask=mask, submask=spot_mask)
+        if within and _check_overlapping_masks(labeled_mask, spot_mask):
+            sample_num += 1
+            # Label spots with unique integers
+            labeled_mask[np.where(spot_mask > 0)] = sample_num
+        if tries > num_masks * 100:  # Prevent infinite loop
+            # warn if stuck, break loop
+            warn("Too many iterations. Placed " + str(sample_num) +
+                 " circular masks instead of " + str(num_masks))
+            break
+    return labeled_mask
+
+
+def _check_overlapping_masks(labeled_mask, new_mask):
+    """
+    Check for any overlapping masks
+
+    Parameters
+    ----------
+    labeled_mask = numpy.ndarray, the labeled_mask that the new_mask may be added to
+    new_mask     = numpy.ndarray, the new proposed region to mask
+
+    Returns
+    -------
+    Boolean, True if new_mask does not overlap the labeled mask
+    """
+    # set all labeled mask values to 1 for bin_mask
+    bin_mask = np.copy(labeled_mask)
+    bin_mask[np.where(bin_mask > 0)] = 1
+    new_bin = np.copy(new_mask)
+    new_bin[np.where(new_bin > 0)] = 1
+    # check max of sum of binary mask and new_mask
+    return np.max(np.add(bin_mask, new_bin)) == 1
+
+
+def _make_random_circle(img, mask, radius=5):
+    """
+    Make a circular ROI at a random point in a mask
+
+    Parameters
+    ----------
+    img    = numpy.ndarray, input image
+    mask   = numpy.ndarray, binary mask of an object
+    radius = int, radius of circular mask to make. Defaults to 5.
+
+    Returns
+    -------
+    spot = PlantCV.Objects class, Circular ROI in random part of mask.
+    """
+    # Find coordinates in mask where mask is non-zero
+    coords = np.column_stack(np.where(mask > 0))
+    # Randomly select a center point from these coordinates
+    center = coords[random.randint(0, len(coords) - 1)]
+    x, y = center[1], center[0]
+    # Create an ROI from the random center point
+    spot = circle(img=img, x=x, y=y, r=radius)
+    return spot
+
+
+def _is_mask_within(full_mask, submask):
+    """
+    Check if a mask is wholly within another mask
+
+    Parameters
+    ----------
+    full_mask  = numpy.ndarray, complete binary mask of an object
+    sub_mask   = numpy.ndarray, binary mask of a smaller part of the full mask
+
+    Returns
+    -------
+    within = Boolean, comparison of full_mask against full_mask and sub_mask.
+    """
+    combined = cv2.bitwise_and(submask, full_mask)
+    within = np.array_equal(combined, submask)
+    return within
diff --git a/tests/plantcv/test_sub_mask.py b/tests/plantcv/test_sub_mask.py
new file mode 100644
index 000000000..c4f3fc626
--- /dev/null
+++ b/tests/plantcv/test_sub_mask.py
@@ -0,0 +1,26 @@
+import cv2
+import numpy as np
+from plantcv.plantcv.submask import sub_mask
+
+def test_sub_mask_success(test_data):
+    """Test for PlantCV."""
+    img = cv2.imread(test_data.small_rgb_img, -1)
+    mask = cv2.imread(test_data.small_bin_img, -1)
+    spots = sub_mask(img, mask, 2, 2)
+    assert len(np.unique(spots)) == 3
+
+
+def test_sub_mask_too_large(test_data):
+    """Test for PlantCV."""
+    img = cv2.imread(test_data.small_rgb_img, -1)
+    mask = cv2.imread(test_data.small_bin_img, -1)
+    spots = sub_mask(img, mask, 2, 20)
+    assert len(np.unique(spots)) == 1
+
+
+def test_sub_mask_too_many(test_data):
+    """Test for PlantCV."""
+    img = cv2.imread(test_data.small_rgb_img, -1)
+    mask = cv2.imread(test_data.small_bin_img, -1)
+    spots = sub_mask(img, mask, 5, 3)
+    assert len(np.unique(spots)) == 2