Recently, the range of the woodland deer mouse (Peromyscus maniculatus gracilis) in Michigan's Lower Peninsula (LP) has contracted simultaneously with the increasing abundance of the white-footed mouse (Peromyscus leucopus). The known LP range of P. m. gracilis has shrunk to two areas, one in Cheboygan/Otsego Counties and another in Alpena County. We used a Geographic Information System habitat analysis to predict where P. m. gracilis could be found between those areas and trapped at selected locations. P. leucopus was present at every location, but the known range of P. m. gracilis had expanded only slightly, and remains as two separate fragments. We analyzed 11 microsatellite loci for all P. m. gracilis to investigate the genetic structure of their remaining LP populations. Genetic diversity was high, F_ST values were low, and there was no evidence of recent bottlenecks, indicating that LP populations of P. m. gracilis once had extensive gene flow across the entire region. However, trapping data, assignment tests, and the distribution of private alleles show that the Alpena population is genetically distinct. We suggest the discordance between analyses could be a sensitive indicator of a population beginning to be affected by isolation, allowing genetic effects of range contractions to be detected in small mammal populations before they become seriously endangered.