We detected 142 six-base restriction sites and 30 haplotypes using 34 enzymes in a survey of mitochondrial DNA (mtDNA) variability in rainbow trout (Oncorhynchus mykiss) from various hatchery sources in Ontario and New York state, and one naturalized population in Lake Ontario. Five major phylogenetic groupings were identified, with the most divergent grouping (assemblage B) differing on average by 0.63% site divergence from other haplotypes; the two most divergent haplotypes differed by 15 site changes. All haplotypes detected in steelhead occur in the phylogenetic assemblage most closely related to chinook salmon (O. tshawytscha). The New York hatchery stocks have markedly lower mtDNA variability than the naturalized population from Ontario. Similarly, the Ontario and New York fish appear to have reduced mtDNA diversity relative to western fish. Significant heterogeneity in mtDNA haplotype frequencies was observed among most Ontario and New York populations examined. Some mtDNA haplotypes were unique to specific populations suggesting their utility as potential markers for genetic stock identification.