Introduction: Sexual dimorphism has attracted the attention of researchers since ancient times and does not always conform to accepted norms. Spix cavies (Rodentia) have been used as experimental models for studies on reproductive biology. This study documented, for the first time, the morphological patterns of differentiation of male and female genital organs and immuno-localized expression steroidogenic enzymes involved in the synthesis of estrogen and androgen hormones throughout embryonic and fetal development of Spix cavy. Material and Methods: Conceptuses were used for anatomical studies, using histological and ultrastructural analyses, and immunohistochemistry for expression of the enzymes cytochromes P450 aromatase (P450arom) and 17a-hydroxylase/17, 20-lyase (P450c17) throughout pregnancy. Results: Undifferentiated gonads of Spix cavy develop into ovaries in females after 25 days of gestation (DG). Ovaries exhibit positive immunoreactivity for P450 arom suggesting the capacity for estrogen synthesis even during fetal development. After 25 DG, paramesonephric ducts develop and differentiate, establishing the oviducts, uterine horns, uterine body and cranial portion of the vagina. The caudal portion of the vagina originates from the urogenital sinus, and the vaginal closure membrane is a result of maintenance of the urogenital membrane. In addition, by 30 DG, the clitoris was flattened with urethral pleats and canalization of the clitoris by the urethra was complete after 40 DG. In males, undifferentiated gonads differentiate into testes by 25 DG. The testis displays immunoreactivity for P45c17, the enzyme responsible for androgen synthesis. Mesonephric ducts differentiate into the epididymis and ductus deferens. The pelvic urethra develops from the urogenital sinus after 25 DG, by folding of the urethral pleats and the complete canalization of the penis by the urethra occurs after 30 DG. Conclusions: Unusual patterns of sexual differentiation was found and further studies are required in order to explain sexual hormones that participate in the complex formation of an external genital appendix in females of Spix cavies. [ABSTRACT FROM AUTHOR]