The salmonids of Pacific North America are a group of closely related species with complex life histories and interesting distribution. Previous studies of their evolution and population structure have involved classical morphological and genetic techniques. We have analysed both intra- and inter-specific variation in mitochondrial DNA sequences of the five North American species of the genus Oncorhynchus and the rainbow trout species, Salmo gairdneri. Cleavage sites for 13 different restriction enzymes were sampled, comparing an average of 48 sites per individual, or approximately 1.7% of the genome. No obvious size variation in the 16 500 ± 500 base pair length was observed. Levels of intraspecific variation detected in the chum salmon and rainbow trout were 0.24 ± 0.23 and 0.45 ± 0.26%, respectively. This variation was population specific; no variation was detected within any of the populations sampled, suggesting the existence of population substructuring. Estimates of divergence between species range from 2.46 ± 0.72% in the coho–chinook salmon comparison to 6.88 ± 1.27% between coho and chum salmon. The phylogenetic relationship among these species, based on the levels of sequence divergence, organizes the species into three distinct groups. One group includes the pink and chum salmon while a second group contains the coho and chinook salmon, as well as the rainbow trout. The sockeye salmon are distinct from both groups. Although most of these results are in accordance with classical analyses, the relationship of the rainbow trout to the coho and chinook salmon suggests different interpretations of the evolution of life histories and morphological traits in these closely related species.