Mitochondrial DNA variation was surveyed among wild anadromous and nonanadromous Atlantic salmon, Salmo salar, which occur in sympatry in Gambo River, Newfoundland. Seventy-one salmon were screened with 18 restriction enzymes, 5 of which revealed nucleotide sequence variation. Nucleotide sequence divergence estimates among the four distinct genotypes detected ranged from 0.2 to 1.0%. Significant heterogeneity in mtDNA genotype frequencies supports the view that exchange of breeding females between groups is infrequent or does not occur, an inference consistent with the observation that the forms use separate spawning sites within the system. Mitochondrial DNA diversity was somewhat greater among the nonanadromous salmon than the anadromous salmon (nucleon diversity (h) = 0.37 and 0.52, respectively). Potential mechanisms for maintaining reproductive isolation include selection of different spawning habitats, different spawning times, and sexual selection based upon the size difference between the two forms.