DNA sequences from the mitochondrial cytochrome-b gene were examined in individuals of the hispid cotton rat (Sigmodon hispidus eremicus) collected from an isolated population in southwestern Arizona. Analyses indicated unique DNA substitutions and haplotypes that were restricted to the population in Arizona and an overall genetic affiliation to populations of the western lineage of S. hispidus, particularly to the population from Otero County, New Mexico. Estimates of time since divergence indicated an allopatric relationship with other populations of S. hispidus ≥150,000 years ago. Additionally, data obtained from analyses of amplified fragment-length polymorphisms indicated that S. h. eremicus exhibits lower genetic diversity compared to other populations. Together, these data along with detection of two replacements of apomorphic nonsynonymous amino acids and genetic divergence among amplified length polymorphisms argue that S. h. eremicus is unique from other populations of S. hispidus and that conservation considerations are encouraged.