Most mammalian ovarian follicles contain only a single oocyte having a single nucleus. However, two or more oocytes and nuclei are observed within one follicle and one oocyte, respectively, in several species, including cotton rat (CR, Sigmodon hispidus). The present study compared ovarian histology, focusing on folliculogenesis, between two inbred CR strains, HIS/Hiph and HIS/Mz. At 4 weeks of age, ovarian sections from both the strains were analyzed histologically. Multi-oocyte follicles (MOFs) and double-nucleated oocytes (DNOs) were observed in all stages of developing follicles in HIS/Hiph, whereas HIS/Mz had MOFs up to secondary stages and lacked DNOs. The estimated total follicles in HIS/Mz were almost half that of HIS/Hiph, but interstitial cells were well developed in HIS/Mz. Furthermore, immunostaining revealed no clear strain differences in the appearance of oocytes positive for Ki67, PCNA, and p63 in MOF or DNOs; no cell death was observed in these oocytes. Ultrastructural analysis revealed more abundant mitochondrial clouds in oocytes of HIS/Hiph than HIS/Mz. Thus, we clarified the strain differences in the CR ovary. These findings indicate that early events during folliculogenesis affect the unique ovarian phenotypes found in CRs, including MOFs or DNOs, and their strain differences.