Interspecific hybridization between Atlantic salmon and brown trout is well documented, but why it should vary so much among populations is not clear. Determining the maternal origin of hybrids can provide insights into the mechanisms underlying interspecific hybridization, but this information is lacking in many studies. Here we present a species-specific mitochondrial DNA marker for the identification of the maternal origin of hybrids. This marker involves only one PCR step followed by fragment analysis, can be integrated within PCR multiplexing for existing nuclear markers for hybrid identification, and is therefore faster and more cost-effective than previous methods.