Stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst) threats world wheat production. Resistance to stripe rust is often overcome by pathogen virulence changes. To unravel the genomic basis of variation and identify avirulence genes in Pst, 30 mutants were developed using ethyl methanesulfonate (EMS) mutagenesis. The progenitor isolate was sequenced, assembled, and annotated for a high-quality reference genome. The mutants were phenotyped for virulence changes on 18 wheat Yr genes for stripe rust resistance and were sequenced. The sequences were mapped to the wild-type reference genome to identify genomic changes. After selecting EMS preferred CG to TA mutations, 217,775 single nucleotide polymorphisms (SNPs) per isolate on average were detected. Among the 30 mutant isolates, 15 changed from avirulent to virulent and the other 15 remained avirulent to YrSP. Association analysis identified 109 SNPs significantly (P < 0.0001) associated with the avirulent and virulent phenotypes to YrSP. Two of the SNPs were responsible for nonsense mutation and 12 SNPs were accountable for missense mutations in the coding regions. Fourteen genes containing non-synonymous mutations were significantly associated to the changes from avirulence to virulence to YrSP. The candidates for the YrSP-avirulence gene and other avirulence genes are valuable for functional confirmation and characterization to unravel the molecular basis of the wheat-Pst interactions.