The displacement loop and NADH-1 dehydrogenase regions of mitochondrial DNA (mtDNA) were amplified by the polymerase chain reaction in 954 Atlantic salmon and digested with 40 restriction endonucleases. Variation was detected with 10 enzymes, resulting in 21 composite haplotypes which were strongly patterned geographically with a major discontinuity observed between most North American (NA) and European salmon. Significant heterogeneity of haplotype frequencies was found within and among all classification levels (continent, country, and river). Haplotype frequencies were significantly different across continents, within European samples, within NA samples, within Canadian samples, within wild Maine samples, within captive Maine strains, and between captive and wild Maine strains. Nine haplotypes occurred only in NA, seven in Maine, three only in Maine, and 11 occurred only in Europe. Some Maine rivers had only a single haplotype, suggesting that effective population sizes may be low. The second most frequent European haplotype occurred in tributaries to one Newfoundland river. Gene trees based on parsimony and genetic distance suggest that the haplotypes are monophyletic within each continent, and that the haplotype found on both continents is intermediate between those of Europe and NA, suggesting common ancestry of all haplotypes.