Genetic markers play an important role in elucidating taxonomic uncertainties for a wide range of organisms. We present a set of specific primers to distinguish between Canis mitochondrial DNA (mtDNA) of New World (North American) and Old World (Eurasian) origin using the ATP-8 region and gel electrophoresis. We amplified mtDNA from Old World (gray wolves [Canis lupus L., 1758]) and New World canids (coyotes [C. latrans Say, 1823] and eastern wolves [C. lycaon Schreber, 1775 or C. lupus lycaon]) collected during 2003–2009 in Québec, Canada, using a multiplexed primer triplet. The results showed a standard band of 150 base pairs (bp) for New World and Old World mtDNA. In addition, Old World mtDNA displayed a second band of 100 bp. The range extent of wolves with New World mtDNA has important implications for canid conservation. The new method can assist conservation managers with rapid and cost-effective screening to monitor 1) the distribution and abundance of wolves with New World and Old World mtDNA, and 2) wolf–coyote hybridization, when used in combination with morphological information and other nuclear markers. © 2012 The Wildlife Society.