Puccinia striiformis f. sp. tritici (Pst) is adept at overcoming resistance in wheat cultivars, through variations in virulence in the western provinces of China. To apply disease management strategies, it is essential to understand the temporal and spatial dynamics of Pst populations. This study aimed to evaluate the virulence and molecular diversity of 84 old Pst isolates, in comparison to 59 newer ones. By using 19 Chinese wheat differentials, we identified 98 pathotypes, showing virulence complexity ranging from 0 to 16. Associations between 23 Yr gene pairs showed linkage disequilibrium and have the potential for gene pyramiding. The new Pst isolates had a higher number of polymorphic alleles (1.97), while the older isolates had a slightly higher number of effective alleles, Shannon's information, and diversity. The Gansu Pst population had the highest diversity (uh = 0.35), while the Guizhou population was the least diverse. Analysis of molecular variance revealed that 94% of the observed variation occurred within Pst populations across the four provinces, while 6% was attributed to differences among populations. Overall, Pst populations displayed a higher pathotypic diversity of H > 2.5 and a genotypic diversity of 96%. This underscores the need to develop gene-pyramided cultivars to enhance the durability of resistance.