Fish assemblages were sampled from streams in Mississippi (fall 2000–2003) allowing a comparison of fish species diversity across drainages and years. Because species diversity is dependent on sample size, we used rarefaction to compare richness and evenness across drainages at a standardized abundance. Rarefied richness was higher in northern drainages (Tennessee, Tombigbee, Lower Mississippi North) than in southern drainages (Pearl, Pascagoula, Lower Mississippi South). Rarefied evenness did not differ across drainages. Diversity and species composition at localities were relatively similar across time. Local diversity was not associated with regional diversity, supporting the idea that local ecological factors limit the number of coexisting species. Higher diversity of the Lower Mississippi North, Tennessee, and Tombigbee drainages is strongly related to their biogeographical history.