The Jico crested-tailed mouse (Habromys simulatus) is an arboreal rodent endemic to Mexico, with only 2 known extant populations restricted to montane cloud forests in the states of Hidalgo and Oaxaca. The habitat of this species has been reduced and fragmented as a result of anthropogenic activities, isolating populations and likely decreasing their sizes. We evaluated the effects of such isolation by analyzing the genetic diversity and structure of H. simulatus using 10 microsatellite loci. DNA was obtained from 52 samples from both populations. Despite the isolated and fragmented nature of the species, we found high levels of genetic diversity (H_Nei  =  0.732), similar to those reported in other endangered species with fragmented distributions. Genetic differentiation was significant (F_ST  =  0.178) and number of migrants was negligible (NmF_ST  =  0.196), a result supported by an assignment test and a factorial correspondence test. Molecular analysis of variance showed that 82% of genetic variation was distributed within populations, not unexpected given that each of the individuals' genotypes was distinct. Individuals within each population were mostly unrelated. The smallest population showed evidence of genetic bottleneck. We found evidence of detrimental genetic processes such as allelic fixation, genetic drift, and inbreeding. Our results strongly suggest that each of the 2 populations is a unique genetic entity that must be considered a distinct evolutionary unit. Unfortunately, both populations are at high risk of extinction, primarily due to habitat loss and population decline.