Examination of variation at polymorphic microsatellite loci is a widely accepted method for determining parentage and examining genetic diversity within rainbow trout (Oncorhynchus mykiss) breeding programs. Genotyping costs are considerable; therefore, we developed a single-step method of co-amplifying twelve microsatellite loci in two hexaplex reactions. The protocol is explicitly described to ensure reproducible results. I applied the protocol to samples previously analyzed at the National Center for Cool and Coldwater Aquaculture (NCCCWA) with previously reported marker sets for a comparison of results. Each marker within the multiplex system was evaluated for duplication, null alleles, physical linkage, and probability of genotyping errors. Data from four of the 12 markers were excluded from parental analysis based on these criteria. Parental assignments were compared to those of a previous study that used five independently amplified microsatellites. Percentages of progeny assigned to parents were higher using the subset of eight markers from the multiplex system than with five markers used in the previous study (98% vs. 92%). Through multiplexing, use of additional markers improved parental allocation while also improving efficiency by reducing the number of PCR reactions and genotyping runs required. I evaluated the methods further through estimation of F-statistics, pairwise genetic distances, and cluster analysis among brood-years at the NCCCWA facility. These estimates were compared to those from nine independently amplified microsatellites used in a previous study. Fst metrics calculated between brood-years showed similar values of genetic differentiation using both marker sets. Estimates of individual pairwise genetic distances were used for constructing neighbor-joining trees. Both marker-sets yielded trees that showed similar subpopulation structuring and agreed with results from a model-based cluster analysis and available pedigree information. These approaches for detecting population substructure and admixture portions within individuals are particularly useful for new breeding programs where the founders' relatedness is unknown. The 2005 NCCCWA brood-year (75 full-sib families) was challenged with Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD). The overall mortality rate was 70%, with large variation among families. Resistance to the disease was assessed by monitoring post-challenge days-to-death. Phenotypic variation and additive genetic variation were estimated using mixed models of survival analysis. The microsatellite markers used were previously isolated from BAC clones that harbor genes of interest and mapped onto the rainbow trout genetic linkage map. A general relationship between UBA gene sequence types and MH-IA-linked microsatellite alleles indicated that microsatellites mapped near or within specific major histocompatibility (MH) loci reliably mark sequence variation at MH genes. The parents and grandparents of the 2005 brood-year families were genotyped with markers linked to the four MH genomic regions (MH-IA, MH-IB, TAP1, and MH-II) to assess linkage disequilibrium (LD) between those genomic regions and resistance to BCWD. Family analysis suggested that MH-IB and MH-II markers are linked to BCWD survivability. Tests for disease association at the population level substantiated the involvement of MH-IB with disease resistance. The impact of MH sequence variation on selective breeding for disease resistance is discussed in the context of aquaculture production.