Mitochondrial DNA (mtDNA) from three Gulf of Alaska and two Bering Sea populations of coho salmon (Oncorhynchus kisutch) was assayed with 21 restriction endonucleases. A restriction site map was constructed for 15 enzymes that recognized hexanucleotide sequences and aligned to the rainbow trout (Oncorhynchus mykiss) gene map using the restriction map and known coho salmon sequences. The restriction site map and gene order of the coho salmon mtDNA genome are consistent with those of rainbow trout. Variation was observed for six enzymes at 10 sites that resulted in eight haplotypes. Variability and divergence observed exceeded those previously observed for allozyme loci in coho salmon. When variability that occurred only in single individuals was eliminated, two haplotypes that differed by four restriction sites remained. Variation at these sites was confirmed from polymerase chain reaction (PCR) amplified fragments. Bering Sea populations exhibited more variation than Gulf of Alaska populations. Explanations for the differences include more recent colonization by or smaller sizes of Gulf of Alaska populations.