Rivers and streams are among the most threatened ecosystems worldwide, and their fish assemblages have been modified by anthropogenic habitat alteration and introductions of non-native species. Consequently, two frequently observed patterns of assemblage change over time are species loss and biotic homogenization. In the present study, we compared contemporary (2006–2007) and historical (1948–1955) assemblages of darters, a group of small benthic fishes of the family Percidae, in the Arkansas River drainage of northeastern Oklahoma, USA. Results showed species loss between the two sampling periods, with historical estimates of overall species diversity across the study area exceeding contemporary estimates by five to eight species. Assemblages showed a low degree of darter similarity based on species presence and absence, with pairwise site comparisons (Jaccard's similarity index) between historical and contemporary samples averaging < 0.35. No significant homogenization or differentiation of assemblages occurred. Range expansion of widespread species, one of the primary mechanisms of biotic homogenization, was not observed; rather, all species occurred at a smaller proportion of sites in contemporary samples. Our results highlight the threat posed by anthropogenic habitat alteration to taxonomic groups such as darters, most of which are habitat specialists. However, our results suggest that biotic homogenization is unlikely to occur in the absence of immigration, especially if assemblages are subjected to ‘novel disturbances’ such as dam construction and watershed-scale habitat degradation which negatively affect all components of the assemblage.