Sequence comparisons were made from 2214 bp of mitochondrial DNA cloned from six Pacific salmonid species. These sequences include the genes for ATPase subunit 6, cytochrome oxidase subunit 3, NADH dehydrogenase subunit 3, NADH dehydrogenase subunit 4L, tRNA(GLY), and tRNA(ARG). Variation is found at 338 silent and 12 nonsilent positions of protein coding genes and 10 positions in the two tRNA sequences. A single 3-bp length difference was also detected. In all pairwise comparisons the sequence divergence observed in the fragment was higher than that previously predicted by restriction enzyme analysis of the entire molecule. The inferred evolutionary relationship of these species is consistent between methods. The distribution of silent variation shows a complex pattern with greatly reduced variation at the junctions of genes. The variation in the tRNA sequences is concentrated in the DHU loop. The close relationship of these species and extensive sequence analyzed allows for an analysis of the spectrum of substitutions that includes the frequencies of all 12 possible substitutions. The observed spectrum of substitutions is related to potential pathways of spontaneous substitution. The salmonid sequences show an extremely high ratio of silent to replacement substitutions. In addition the amino acid sequences of the four proteins coded in this fragment show a consistently high level of identity with the Xenopus sequences. Taken together these data are consistent with a slower rate of amino acid substitution among the cold-blooded vertebrates when compared to mammals.