Variation in habitat across the landscape can limit dispersal between populations and influence the diversity and degree of genetic structure of populations. This effect may be larger in small aquatic species with low vagility, specific habitat requirements, and that inhabit lotic systems where dispersal may be biased by the unidirectional movement of water downstream. Darters offer a unique perspective to understand these influences because they have variable life histories and habitat requirements, and evidence suggests that certain life history characters may affect gene flow. Data were collected from a combination of microsatellite loci from 825 individuals to compare genetic structure and differentiation of three species of darters (Ammocrypta beanii, Etheostoma swaini, and Percina nigrofasciata) with differences in life histories and habitat requirements in the Pearl River basin. Our analyses reveal low, but interesting, patterns of population subdivision. Genetic subdivision among populations was highest for E. swaini, intermediate for P. nigrofasciata, and lowest for A. beanii. This pattern suggests a difference in dispersal, influenced by the variation in available habitat required for each species. Further investigations are warranted for small, benthic, and especially threatened species in the Pearl River basin as the main channel has undergone a significant shift in geomorphology and habitat diversity since the construction of reservoirs and low-head dams along its course.