Natural hybridization between pink salmon (Oncorhynchus gorbuscha) and Chinook salmon (O. tshawytscha) has been observed only in the Great Lakes, where both species have been introduced. The direction of hybridization between these species is poorly understood, thus the present study analyzed mitochondrial DNA (mtDNA) to determine whether the maternal parents were pink or Chinook salmon. During annual salmon population surveys on the St. Marys River (1998–2002), fifty putative hybrids of pink salmon and Chinook salmon were identified from meristic, morphometric and color characters. We designed primers to amplify a fragment of the mitochondrial D-loop control region that included both a variable and a control restriction site (BstNI). Polymerase chain reaction, restriction endonuclease digestion and capillary electrophoresis of mtDNA were used in order to identify the maternal parent of each hybrid salmon. In addition, the amplified fragments from the three fish species were sequenced to further verify the results. All hybrid salmon specimens were found to possess Chinook salmon mtDNA, indicating that hybridization between Chinook salmon and pink salmon is asymmetrical and likely unidirectional, occurring only between male pink salmon and female Chinook salmon. Influences contributing to the hybridization of these salmonid species could include limited spatial and temporal segregation of spawners, sexual selection, a limiting number of Chinook males, and/or physiological factors.