A 60 bp sequence variation hotspot in the canine mitochondrial DNA hypervariable region 1 was evaluated for its use in forensic investigations. Nineteen haplotypes containing 18 single nucleotide polymorphisms were observed among laboratory-generated and GenBank-derived domestic dog sequences representing five regional localities in the U.S. Samples from the different localities were highly variable with the levels of intra-population variability being similar among the populations studied. AMOVA further confirmed that there was no significant genetic structuring of the populations. Assays using these haplotypes were robust, canid specific and portend a rapid method for correctly excluding individual dogs as noncontributors of forensic evidence. Species-specificity of the primers was confirmed by means of in-tube polymerase chain reaction of human and cat DNA and in-silico assessment of the genomes of several animal species. Breed-specific fragments were not detected among the common haplotypes but there is evidence that this assay may be capable of differentiating domestic dog, wolf, and coyote sequences.