Cotton (Gossypium hirsutum L.) is utilized commercially in the production of textile, clothing, and household goods. Its growth is affected by various environmental conditions such as soil, climate, and water supply. Irrigation is one of the most important factors for crop management. This research was designed with the aim of studying the biochemical and anatomical features of two genotypes of cottonseed (Latif and Golestan) in order to determine optimum irrigation. Protein, starch, gossypol gland, total soluble protein, starch content, and the anatomical structure of the cotton seeds were investigated. The data were analyzed using the SAS Software. The results showed that when rainfed, Golestan genotype seeds had a larger number of secretory cavities, soluble protein, and starch compared to Latif seeds. There was also a noticeable difference in the size of the seeds. Cellulosic cell walls and protein particles were also observed in the seed structure under induced water stress conditions. It can therefore be concluded that in response to water shortage, there was a noticeable change in the morphometric, anatomic, and biochemical features of the cottonseeds. It can be concluded that when rainfed, the Golestan genotype of cottonseed has more compatibility than the Latif genotype. In general, the application of different levels of irrigation showed that at moderate levels of irrigation, anatomical features appeared more normal. The observations indicate that at high levels of irrigation, some cells begin to dehisce due to osmotic stress, which results in a lack of accurate formation of tissue structures. The Golestan genotype is therefore the best suited for dryland farming.