Freshwater mussels (unionids) are increasingly recognized as important providers of ecosystem services, yet are among the most endangered fauna in the world. Because unionids are generally sessile and require specific fish hosts for development and dispersal, they are particularly vulnerable to habitat degradation. Surprisingly, little is known about the distribution of genetic diversity in freshwater mussels and this gap has a negative impact on taxonomy, monitoring, conservation and ecological research in these species. Here, we focus on western North American Anodonta, one of only three genera known to exist in this broad landscape and which contains three highly divergent lineages. We describe phylogeographical subdivision in the most widespread and diverse of these lineages, which includes Anodonta californiensis and Anodonta nuttalliana and occurs from Canada to Mexico. Using mitochondrial and nuclear data, we found that genetic structuring within this clade is inconsistent with morphologically based species designations, but instead follows patterns of vicariance among major hydrogeologic basins. Furthermore, there was a strong tendency for population diversity within drainage systems to increase downstream, implying greater habitat or host fish availability in this direction. Microsatellite results indicated that sampling locations were all genetically distinct, even at short distances. Many of our sample populations showed evidence of a recent demographic bottleneck, although this effect seemed to be very local and not drainage or basin-specific. This study provides a foundation for the establishment of appropriate management units and future research on adaptive differentiation and host fish relationships.