A collection of natural-origin spring Chinook from 1986 was compared to samples from two spawner groups (supplementation program and in-river spawners), and to collections of hatchery- and natural-origin from the Tucannon River. Samples from the captive brood program at the Tucannon River Hatchery were also compared. A microsatellite DNA analysis was conducted to determine if there have been any changes to the genetic diversity of spring Chinook in the Tucannon River. The measures of genetic diversity (heterozygosity and allelic richness) revealed similar levels within each spawner group and collection based on origin over time. Assessment of within population diversity indicates that the spawner groups and collections by origin have not undergone a loss of diversity and are not represented by family groups. We did detect that collections of the captive brood are not within Hardy-Weinberg proportions and have significant linkage disequilibrium as a possible result of using equal numbers of individuals from two brood years that are differentiated. The collection of captive brood progeny returns in 2008; however is within expected proportions and indicates there has not been a genetic change to the spawner group collection or collections by origin. The pairwise FST values identify the variation between any two groups is approximately 1.0% or less indicating the differences among the groups is small. Factorial correspondence analysis identifies similarity among collections that are separated by four years and represent the genetic differences among primary brood years and not genetic changes to the natural-origin collection from 1986. The combination of all the results demonstrates that the genetic diversity of spring Chinook in the Tucannon River has not significantly changed as a result of the supplementation or captive brood programs.