Spermatogenesis, the production of sperm, is a fundamental part of the reproductive cycle in vertebrates and warrants detailed study. Reptiles exhibit a hybrid pattern of spermatogenesis that has similarities with both anamniotes and amniotes. Researching this interesting transition will help us better understand vertebrate evolution. To this end, we histologically examined the spermatogenic and testicular cycle of Rough Greensnakes (Opheodrys aestivus) from Arkansas. We hypothesized that spermatogenesis in Rough Greensnakes would follow the different spermatogenic stages found in previously published studies on temperate snakes. In addition, we hypothesized that both season and seminiferous tubule diameter would have a significant effect on seminiferous tubule epithelial height. Finally, we hypothesized that Rough Greensnakes would exhibit postnuptial spermatogenesis. We constructed a cell wheel illustrating the chronological development of all germ cell stages for this species and measured seminiferous tubule diameter and seminiferous tubule epithelial height from 22 specimens. Opheodrys aestivus exhibited small seminiferous tubule diameters in spring followed by an increase in summer. By October the lumen was mostly empty of sperm because they had migrated to the vas deferens for winter storage. Seminiferous tubule epithelial height was significantly correlated with seminiferous tubule diameter. Both seminiferous tubule diameter and season significantly affected seminiferous tubule epithelial height. This species can be categorized as having postnuptial spermatogenesis where sperm are produced in the summer after spring mating. Future studies of O. aestivus need to investigate the testicular cycle in other parts of their geographic distribution to see if this monthly pattern is consistent.