Understanding of long-term virulence dynamics of pathogen populations in response to host resistance gene deployment is of major importance for disease management and evolutionary biology. We monitored the virulence dynamics of Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, over 25 years in France. Virulence dynamics was explained by estimates of area associated with resistance genes carried by farmers' cultivars. The epidemics assessed through disease severity significantly correlated with the number of P. striiformis f. sp. tritici isolates collected each year, used to describe virulence dynamics. In the south, the dominance of the Mediterranean pathotype 6E16 and the cultivation of a susceptible cultivar were associated with an epidemic from 1997 to 1999. In the north, five epidemics occurred due to successive acquisition of virulence to the resistance genes Yr7, Yr6, Yr9, Yr17, and Yr32, either by acquisition of the virulence in the previous dominant pathotype or by incursion or selection of one or two new pathotypes. Frequency of pathotypes with Vr7 and Vr6 declined with the reduction in the cultivation of corresponding Yr gene cultivars, whereas the virulence Vr9 persisted longer than the cultivation of Yr9 cultivars. Although the first pathotypes carrying Vr9 decreased, this virulence persisted in other pathotypes even in the absence of Yr9 cultivars. At the regional level, Yr9 cultivars in the north caused a shift from high Vr6 frequency to high Vr9 frequency whereas, in the central region, where Yr9 cultivars were rare, Vr6 remained prevalent.