The checkered beetle, Thanasimus dubius F., is an important predator of scolytid bark beetles that attack conifers. Relatively few studies exist that have addressed the population genetics of predatory beetles, especially those with potential as biological control agents. This study was conducted to investigate the population genetics of T. dubius across a large part of its range in the eastern United States. A 464-base pair portion of the mitochondrial cytochrome c oxidase subunit I was sequenced for 85 individuals resulting in 60 haplotypes. Analysis of molecular variance was conducted on the resulting haplotypes for all populations and as a hierarchical analysis between populations defined as broad-scale northern and southern groups. Results indicate a significant overall ΦST = 0.220 (P < 0.001) for all populations with the hierarchical analysis revealing that this significant ΦST is due to structuring of the populations between northern and southern regions (ΦCT = 0.388, P < 0.009). The observed genetic structure is possibly due to the discontinuous distribution of pine trees, which act as hosts for the prey of T. dubius, which has occurred historically in the central region of the United States that has been covered by prairie.