Aim Geomorphic evolution of river basins can shape the structure and diversity of aquatic communities, but understanding the biological significance of basin evolution can be challenging in semi-arid regions with ephemeral or endorheic conditions and complex drainage configurations such as the Sierra Madre Occidental (SMO) in North America. In this study, we characterized range-wide patterns of genetic variation in the Mexican stoneroller (Campostoma ornatum) to infer how orogenic and erosional influences on river basin connectivity have given rise to the diverse and largely endemic freshwater communities across the SMO region.Location Twelve drainage basins across northern Mexico and the south-western United States, centred on the SMO.Methods We collected 202 specimens from 98 localities across the range of C. ornatum. We performed phylogenetic analyses of DNA sequences from one mitochondrial (cytochrome b) and one nuclear (intron S7) gene. Phylogenetic trees were estimated for each data set using maximum likelihood and Bayesian inference.Results Phylogenetic analyses consistently resolved a monophyletic C. ornatum composed of multiple evolutionary lineages within two markedly divergent clades that differentiate northern drainages from southern drainages in the SMO region. Within-clade patterns of divergence corresponded to fine-scale geographic structure within and among SMO drainage basins. However, the geographic distribution of evolutionary lineages within the northern and southern clades did not always correspond to the geographic configuration of drainage basins. Some subclades encompassed multiple drainages, and individuals from a single drainage were sometimes recovered in multiple subclades. Main conclusions Our findings indicate that a common ancestor of MexicanCampostoma is likely to have entered north-west Mexico through an ancient Rio Grande system that extended as far south as the Rio Nazas and Rio Aguanaval. The geographic orientation of the two strongly divergent clades recovered within C. ornatum provides evidence of long-standing isolation of southern basins from northern basins within the ancestral Rio Grande system, possibly due to the combined influence of tectonic events and increasing regional aridity. Geographic patterns of genetic variation also provide evidence of range expansion from Atlantic to Pacific drainages due to drainage evolution and river capture events, as well as further inter-basin exchange via more recent headwater capture events, hydrological connections and possible anthropogenic introductions.

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