SummaryReproductive biology of invasive species is not often studied relative to the invasion process, although it may provide an accurate indicator of the invasion stage. We evaluated the reproductive biology of the exotic fish species Pseudoxiphophorus bimaculatus and Poecilia sphenops in the Teuchitlán River, Jalisco, Mexico by fertility, size at first maturity, sex ratio, gonad maturity stage, gonadosomatic index, condition factor, size-structure, and habitat. The reproductive variables were related to environmental characteristics using the non-metric analysis of multidimensional scaling. A total of 1374 specimens of P. bimaculatus and 571 of P. sphenops were captured by seine netting and electrofishing. Maximum fertility of P. bimaculatus was 15.99 ± 2.27 embryonated eggs and embryos and, for P. sphenops, 31.26 ± 4.17. Females predominated among P. bimaculatus, while in P. sphenops the sex ratio was ~1:1. We found mature female and male of P. bimaculatus in degraded sites and juveniles in the springs. Poecilia sphenops reproduced along the river. The establishment of both invasive species in the Teuchitlán River is evidence that they share the reproductive habitat with native fish species, and tend to spread and colonize new areas.