Despite the increase of exotic species in bodies of water, studies on the population structure of coexisting native and exotic fish species are rare. We evaluated the population structure of the native Poecilia mexicana and non-native Poeciliopsis gracilis species in an environmental gradient and its relation to the habitat characteristics in the Jalpan River, located in the state of Queretaro, Mexico. We calculated size structure, sex ratio, gonad stage, and condition factor for both species. Visual-based habitat quality, riparian quality, and physical and chemical characteristics of water were also evaluated. A total of 322 individuals of P. gracilis and 762 of P. mexicana were captured with different fishing nets due to the habitat heterogeneity. Study sites were classified into three categories of habitat conditions: poor, marginal, and sub-optimal. P. mexicana females showed a complete structure of sizes in the poor habitat category. P. gracilis did not show a complete structure in any habitat category. The sub-optimal category of habitat conditions had the lowest number of specimens for both species. Poor category showed a sex ratio of 1:1 (females: males) for P. mexicana while in P. gracilis, females were more abundant than males (sex ratio 5:1). Fulton's factor was higher for P. mexicana than for P. gracilis. Our results showed that habitat quality of the river affected size structure, stages of development, and sex ratio of the two species, but did not show conclusive evidence of favored species or reproductive competition.