The firefly Microphotus octarthrus occurs allopatricallly throughout pinyon-oak and juniper- oak habitats of the deserts of southwestern US. Adult dispersal is limited thus individuals are likely to be genetically isolated by geographic distances. However, during the Pleistocene, habitats may have been contiguous allowing for greater dispersal and genetic continuity. In this study we investigated the patterns of mitochondrial cytochrome oxidase I haplotypes among disjunct populations of M. octarthrus to determine whether current populations were historically contiguous. A total of 26 mtDNA COI haplotypes was found for 28 individuals from six populations spanning the beetle's range. Cladistic analysis of individuals resulted in 15,500 equally parsimonious trees. Strict consensus of these trees was mostly resolved except for a few individuals within well supported clades. Individuals from the same population were monophyletic and genetic isolation by distance was observed. Mean branch lengths for the clades of individuals from Texas, western New Mexico, and northern New Mexico were 4.4%, 1.6%, and 5.0%, respectively. Given an average sequence divergence of 2.3% per million years, isolation of these groups coincided with the beginning and middle of the Pleistocene. Thus, populations of M. octarthrus and their habitat were likely disjunct throughout the Pleistocene and the dispersal of these beetles was limited. In addition, branch length values were similar to values observed among the other Microphotus species. This suggests the possibility of cryptic species, however limited life history data renders recognition of new species difficult. Pre-mating barriers such as female advertising posture, male approach, and coupling time would further support the isolation of these populations and recognition of new species.