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Yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst), is economically one of the most important diseases of wheat in India and worldwide. During four wheat-growing seasons (2015-19), a total of 951 yellow rust samples collected from farmers' fields (n = 602) and trap plot nurseries (n = 349) were analyzed for virulence on 30 differentials. From this analysis, 11 distinct Pst pathotypes were identified. The virulence pattern of these pathotypes was also tested on 14 Avocet near-isogenic lines (NILs) and Avocet S. None of the pathotypes were virulent to Yr5, Yr10, Yr15 , Yr16, Yr24 , Yr32, and YrSP. Pathotype 46S119 (46E159) was most prevalent and widely distributed in all the areas and its overall frequency was maximum (56.2%) followed by 110S119 (110E159, 31.9%). However, the frequency of 46S119 has decreased gradually from 62.0% (2015-16) to 48.4% (2018-19). Pathotype 238S119 (238E159) being the most virulent among the identified pathotypes, was detected in 6.8% of samples. Each of the remaining pathotypes was identified in <5% of the samples. Genetic diversity among eleven pathotypes was assessed using novel 89 SSR primer-pairs out of which 24 primers showed polymorphism. Finally, 69 alleles were detected across the loci with an average of 2.87 alleles per locus. Polymorphic information content (PIC) value was in the range from 0.28 to 0.68, with a mean of 0.47. Expected heterozygosity was in the 0.34 to 0.73 range, with a mean of 0.56. Cluster analysis based on both avirulence/virulence pattern and SSR profile categorized the identified pathotypes into two major clades. Generated information will potentially be helpful in rust resistance breeding and deployment of resistant cultivars in yellow rust-prone areas. • Yellow (stripe) rust is economically the most important disease of wheat in India. • Disease is caused by a fungus Puccinia striiformis f. sp. tritici (Pst). • Phenotypic and genotypic variability among the Pst pathotypes was explored. • Novel polymorphic microsatellite markers were also developed. • Generated information is prerequisite for rust resistance wheat breeding. [ABSTRACT FROM AUTHOR]