The western Himalayan region in Pakistan has been shown to be the center of diversity of Puccinia striiformis; however, little is known about its genetic relations with the eastern part of the Himalayas. We studied the genetic structure of P. striiformis from Nepal (35 isolates) and Bhutan (31 isolates) in comparison with 81 Pakistani samples collected during 2015 and 2016, through microsatellite genotyping. Genetic analyses revealed a recombinant and highly diverse population structure in Pakistan, Bhutan, and Nepal. A high level of genotypic diversity (>0.90) was observed for the three countries of Pakistan (0.96), Bhutan (0.96), and Nepal (0.91) with the detection of 108 distinct multilocus genotypes (MLGs) in the overall population; 59 for Pakistan, 27 for Bhutan, and 26 for Nepal. Mean number of alleles per locus and gene diversity were higher in Nepal (3.19 and 0.458, respectively) than Bhutan (3.12 and 0.458, respectively). A nonsignificant difference between the observed and the expected heterozygosity in all populations further confirmed the recombinant structure. A clear population subdivision between the Himalayan region of Nepal, Bhutan, and Pakistan was evident, as revealed by FST values (ranging between 0.111 to 0.198), discriminant analysis of principal components, and resampling of MLGs. Limited gene flow could be present between Nepal and Bhutan, while the population from Pakistan was clearly distinct, and no divergence was present between two populations from Pakistan (Bajaur and Malakand). The overall high diversity and recombination signature suggested the potential role of recombination in the eastern Himalayan region (Nepal and Bhutan), which needs to be considered during host resistance deployment and in the context of aerial dispersal of the pathogen. Further surveillance should be made in the Himalayan region for disease management in the region and in the context of worldwide invasions.