No geographic differences in mitochondrial DNA (mtDNA) distribution among United States (US) domestic dog populations have been detected to date. To test the hypothesis that regional differences exist, a 608 bp sequence of the canid mtDNA hypervariable region 1 (HV1) from 220 mixed breed animals from the Western, Northeastern, Midwestern, and Southern US were combined with 429 published mixed and pure breed dog HV1 sequences to form a substantial geographically representative dataset. With an increased sample size of regionally representative sequences, geographic substructure among regional populations was shown to be statistically significant using the modified Fisher's exact test and pairwise Fst. The results of the AMOVA showed that 91% of the variation is present within the regional dog populations. Based on these analyses, the significance of regional canine HV1 haplotype distributions and frequencies demonstrate further the value of regional and mixed breed canine mtDNA in forensic investigations in the US.