Stripe (yellow) rust, caused by Puccinia striiformis f. sp. tritici, is a serious disease of wheat in the world. The obligate biotrophic fungal pathogen changes its virulence rapidly, which can circumvent resistance in wheat cultivars and cause severe epidemics. Because P. striiformis f. sp. tritici races have been identified in the United States using different wheat genotypes in different time periods, it is difficult to make direct comparisons of the current population with historical populations. The objective of this study was to characterize historical populations with 18 Yr single-gene lines that are currently used to differentiate P. striiformis f. sp. tritici races in order to understand virulence and race changes of the pathogen over 40 years in the United States. From 908 P. striiformis f. sp. tritici isolates collected from 1968 to 2009 in the United States, 171 races were identified and their frequencies were determined. More races, more new races, and races with more virulence genes were detected since the year 2000 than prior to 2000. None of the races were virulent to Yr5 and Yr15, indicating that these genes have been effective since the late 1960s. Virulence genes to the remaining 16 Yr genes were detected in different periods, and most of them increased in frequency over time. Some virulence genes such as those to Yr17, Yr27, Yr32, Yr43, Yr44, YrTr1, and YrExp2 appeared 14 to 37 years earlier than previously reported, indicating the greater value of using Yr single-gene lines as differentials. Positive and negative associations were detected between virulence genes. The continual information on virulence and races in the P. striiformis f. sp. tritici populations is useful for understanding the evolution of the pathogen and for breeding wheat cultivars with effective resistance to stripe rust.