Technical Abstract: Knowledge of the genetic diversity existing in previously released hard red winter wheat (HRWW, Triticum aestivum L.) cultivars in the Great Plains region, United States, is essential for effective utilization of these genetic resources in the various HRWW breeding programs. To ascertain a measure of the genetic diversity of the existing US HRWW, 60 cultivars were analyzed with 62 microsatellite markers distributed throughout the wheat genome. Marker data were subjected to distance-based analysis and analysis of molecular variances. In total, 341 polymorphic alleles were scored with a range of 2—12 alleles per locus. Genetic diversity gradually increased in cultivars released after the 1970s. Cultivars released in the 1990s had the highest allelic richness (4.79), gene diversity (0.60), and polymorphic information content (0.56). Levels of genetic diversity were similar between the major HRWW breeding programs. Cluster analysis resulted in eight clusters. Cluster grouping gave close matches with pedigrees and with regional distribution of the cultivars. Using decadal information, cultivars released from 1900—1969 were grouped into one cluster, cultivars from 1990—2005 were grouped into a separate cluster, whereas cultivars from the 1980s did not group with any other decades. Analysis of molecular variance revealed a significant variation among the clusters, signifying that a true genetic variation existed among the clusters. The higher proportion of genetic variation explained by cultivars within clusters compared with among clusters indicates greater genetic diversity among cultivars within clusters. Our results indicate that genetic diversity of Great Plains HRWW cultivars has increased in the past century, and the trend is continuing.