The rapid advance of high-throughput sequencing has prompted a transition in wildlife and fisheries genetics from using microsatellites toward markers that are more amenable to genotyping by sequencing. Microhaplotypes are novel multi-allelic genetic markers that utilize a high-throughput genomic amplicon sequencing approach to genotype large numbers of individuals for parentage and kinship analysis and population genetic studies, including applications in monitoring and fisheries management. We describe the development of a panel of microhaplotypes for Oncorhynchus mykiss, a species of high cultural and economic importance both in its native range in the North American and the Kamchatka Peninsula of northeast Asia, and globally through introductions for aquaculture and due to its reputation as a prized sport fish among recreational fishers. The panel includes 124 loci presumed to be neutral, a marker for the sex determination locus (SdY), and 10 loci targeting previously identified adaptive genomic variants associated with important life-history traits in this species. We demonstrate that this panel provides high resolution for phylogeographic and other genetic analysis and provide an initial standardized reference population genetic baseline of California O. mykiss.