Interior cutthroat trout Oncorhynchus clarkii have undergone severe declines over the past 150 years. Many subspecies now persist in a highly fragmented state, primarily within headwater streams. We used 12 microsatellites to investigate the population genetic characteristics of 22 remnant populations of Rio Grande cutthroat trout O. c. virginalis isolated in montane streams in New Mexico. Populations varied markedly in the amount of genetic diversity they contained. There was no significant relationship between estimated adult population size or habitat size and heterozygosity; however, populations occurring above natural barriers were significantly less diverse. Seven population samples exhibited significant deviations from Hardy-Weinberg equilibrium. Interlocus variance in the population inbreeding coefficient F IS was correlated with habitat size, and several population samples exhibited a significantly higher variance in interindividual relatedness, or a significantly higher median individual inbreeding coefficient, than would be expected by chance. These results suggest that cutthroat trout populations in headwater streams consist of multiple partially discrete subpopulations in which only a small number of adults successfully reproduce. The potential for such population substructure should be considered when planning management activities for stream-dwelling cutthroat trout.