Two major Ovis aries mitochondrial DNA (mtDNA) haplogroups have been described in independent studies. HinfI RFLP data of mitochondrial genomes from a large sample set (n = 239) indicated an ancient mutation which differentiates between the two mtDNA types. A completely determined sheep mtDNA sequence was used to assign this mutation to the COI gene and to develop a PCR based assay discriminating between the two phylogenetic branches. The haplogroup specificity of the mutation was further investigated in 26 randomly selected individuals. The animals were unequivocally assigned to their respective groups on the basis of the developed test and their complete control region sequences. The assay provides a rapid and economic means of discriminating between both major domestic sheep mtDNAs.