Data collected over the past 15 years in Colorado show that deer mice (Peromyscus maniculatus) collected in southeastern Colorado have a significantly lower Sin Nombre virus (SNV) antibody prevalence than mice from western Colorado. Based on mitochondrial sequences, P. maniculatus was recently subdivided into 6 clades. Clade 1a occurs throughout the mountainous regions of western Colorado and clade 2 crosses the short-grass steppe in southeastern Colorado. We used mitochondrial single nucleotide polymorphisms (SNPs) from this earlier study and 14 nuclear SNPs to test the hypothesis that clade 2 mice consist of a separate subspecies. Little genetic differentiation was detected (F_ST  =  0.027) among 435 deer mice collected from 8 locations in western Colorado. In contrast, 171 deer mice collected from 4 locations in eastern Colorado were genetically differentiated from one another (F_ST  =  0.168) and from those captured in western Colorado (F_ST  =  0.256). During this survey we identified a single locality in central Colorado where both clade 1a and 2 mice are sympatric and have a high SNV seropositivity (52.2% in 2006; 24.4% in 2009). Antibody prevalence was 21.1% among clade 2 mice, lower than the 32.4% rate among clade 1a mice, suggesting that clade 2 mice may, for both genetic and environmental reasons, have lower susceptibility to SNV infection.