The Rio Grande cutthroat trout, Oncorhynchus clarkii virginalis, has declined precipitously over the past century, and currently exhibits a highly fragmented distribution within the Canadian, Pecos and Rio Grande river systems of the western United States. The relationships between populations in the three river drainages, and between O. c. virginalis and the closely related taxa O. c. pleuriticus and O. c. stomias, are not well understood. In order to guide management decisions for the subspecies, we investigated the distribution of variation at 12 microsatellite loci and two regions of the mitochondrial genome. We observed a high level of genetic differentiation between O. c. virginalis populations occupying different headwater streams (global F_st = 0.41). However, we found evidence for previous gene flow within the Rio Grande drainage, indicating that inter-population differentiation may have been exacerbated by the recent effects of population fragmentation. Despite large-scale anthropogenic movement of individuals from the Rio Grande into the Canadian and Pecos, the genetic signature of long-term evolutionary independence between the three drainages has been retained.