Barley (Hordeum vulgare L.) is the most important cereal crop in the Qinghai-Tibet Plateau, and the yield has been seriously threatened by Blumeria graminis f. sp. hordei (Bgh) in recent years. To understand the virulence and genetic traits of different Bgh populations, 229 isolates of Bgh were collected from Tibet, Sichuan, Gansu and Yunnan provinces of China during 2020 and 2021, and their pathogenicity to 21 barley lines of different genotypes was assessed. A total of 132 virulent types were identified. The Bgh isolates from Yunnan showed the highest diversity in terms of virulence complexity (Rci) and genetic diversity (KWm), followed by those from Sichuan, Gansu, and Tibet, in that order. Single nucleotide polymorphism (SNP) in genes coding for alternative oxidase (AOX), protein kinase A (PKA), and protein phosphatase type 2A (PPA) were detected at seven polymorphic sites. Nine haplotypes (H1–H9) with an average haplotype diversity (Hd) and nucleotide diversity π of 0.564 and 0.00034, respectively, were observed. Of these, haplotypes H1 and H4 accounted for 88.8% of the isolates, and H4 was predominant in Tibet. Genetic diversity analysis using the STRUCTURE (K = 2) and AMOVE indicated that the inter-group variation accounted for 54.68%, and inter- and intra-population genotypic heterogeneity accounted for 23.90% and 21.42%, respectively. The results revealed the recent expansion of the Bgh population in Tibet, accompanied by an increase in virulence and a loss of genetic diversity.