The penile tubular urethra forms by canalization of the urethral plate without forming an obvious urethral groove in mice, while the urethral epithelium forms a fully open urethral groove before urethra closure through the distal-opening-proximal-closing process in humans and guinea pigs. Our knowledge of the mechanism of penile development is mainly based on studies in mice. To reveal how the fully opened urethral groove forms in humans and guinea pigs, we compared the expression patterns and levels of key developmental genes using in situ hybridization and quantitative PCR during glans and preputial development between guinea pigs and mice. Our results revealed that, compared with mouse preputial development, which started before sexual differentiation, preputial development in guinea pigs was delayed and initiated at the same time that sexual differentiation began.