Summary: To investigate intergender differences in muscle cleavage and joining during development of the external anal sphincter (EAS), we examined semiserial sections of 16 fetuses between 15 and 30 weeks of gestation (6 males and 10 females). The subcutaneous part of the EAS (EASsc) developed along the male perineal raphe and extended posteriorly. Thus, the male EAS was characterized by anterior protrusion of the subcutaneous muscle, in contrast to the almost circular female EAS. In both genders, the bulbospongiosus anlage (or the levator ani anlage) issued muscle fibers to form the superficial (or deep) part of the EAS. The EASsc communicated with the superficial part in males, whereas the female bulbospongiosus tended to communicate with the levator ani rather than the EAS. In both genders, the longitudinal muscle bundle(s) of the anorectum contributed to perineal body formation. However, the male perineal body also had a thick fascia between the rhabdosphincter and the levator. The bulbospongiosus seems to play a critical role in forming the EAS. A strict intergender difference in subcutaneous muscle development is evident along the perineal raphe, as the raphe is not evident in females. These results help to explain variations in the EAS, including anal malformations.