The combination of whole-genome sequencing efforts and emerging high-throughput genotyping techniques has made single nucleotide polymorphisms (SNPs) a marker of choice for molecular genetic analyses in model organisms. This class of marker holds great promise for resolving questions of phylogeny, population structure, introgression, and adaptive genetic variation. Fifty-five polymerase chain reaction primer pairs were used to target variable regions of the rainbow trout Oncorhynchus mykiss genome, 48 of which were designed from information found in publicly available DNA sequence databases. Forty of these primer pairs yielded sequenceable products. These sequences were compared across 1À10 individual fish from each of the following representative subspecies and strains: Sacramento redband trout O. mykiss stonei, California golden trout O. mykiss aguabonita, Little Kern golden trout O. mykiss whitei, coastal rainbow trout O. mykiss irideus, and the Mount Shasta hatchery strain. A total of 208 SNPs were identified in 37 loci, and a range of 75-128 SNPs were observed in pairwise comparisons of any two representative trout groups. As a test of high-throughput genotyping, the TaqMan 5 0 nuclease assay was used to genotype 335 fish representing 14 populations at SNP LDH-156*, enabling us to characterize allelic frequencies in larger sample sizes and additional populations of each subspecies.