To understand the inheritance of the TSA-6 Puccinia striiformis f. sp. tritici (Pst) isolate that is virulent to Yr5 and was recently detected in China, we analyzed avirulence and virulence of 120 selfed progeny lines from Berberis shensiana. The results showed that the TSA-6 isolate is virulent against the Yr5 resistance gene, and overall progeny lines were categorized into 73 virulence phenotypes (VPs); of these, 72 VPs differed from the isolate TSA-6, and only one VP, including three progeny, was identical to the parental isolate. The analyses indicated that the TSA-6 isolate is homozygous for avirulence at the Yr10, Yr15, and Yr26 resistance loci and virulence at the YrA resistance locus. The TSA-6 isolate is heterozygous for avirulence at the Yr2, Yr3, Yr5, Yr7, and Yr8 resistance loci, which are controlled by a dominant/recessive relationship. The Yr1, Yr6, Yr9, Yr17, Yr27, Yr25, Yr28, Yr29, Yr32, YrTr1, and YrSP resistance loci are governed by two complementary dominant/recessive genes. Avirulence against heterozygous Yr4, Yr43, Yr44, Yr76, and YrExp2 resistance loci is regulated by a dominant and recessive or a dominant and suppressor gene pair. In total, 117 multilocus genotypes were detected at 24 KASP-SNP marker loci among the 120 progenies. Using these marker loci, we constructed a linkage map with a genetic distance interval spanning 624.5 cM. Quantitative trait loci corresponding to phenotypic segregation for virulence at 20 Yr resistance loci in addition to the Yr1 resistance locus were identified. These results facilitate our understanding of Pst virulence evolution and simplify breeding of wheat cultivars with effective resistance to wheat stripe rust.