Gossypol is a terpenoid aldehyde found in cotton (Gossypium hirsutum L.) glands that are located throughout the plant and seed, where it serves a protective function against pests and pathogens. Cottonseed use is limited mainly to cattle feed because gossypol is toxic to most animals except ruminants. Lowering gossypol content in the seed would increase the possible uses for cottonseed. Developing new strategies to modify gossypol in cottonseed requires a better understanding of the development of gossypol containing glands. The first step is to determine when gossypol glands are initiated and filled with gossypol. Gland development was investigated using microscopic images of developing seeds from 10 glanded and two glandless cotton lines. A digital microscope with a VH-Z20R (20X to 200X) lens was used to capture developing ovule (seed) images at 14 to 22 d after flowering (DAF). One boll per plot was imaged for each DAF time point and five different sets of time intervals were collected in each of two years. Imaging revealed empty glands forming as early as 14 DAF and as late as 20 DAF. For most of the entries, some glands were filling by 18 DAF and as early as 16 DAF for ultra early lines (< 110 d to maturity).