Bacterial leaf blight (BLB) disease of rice caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the significant constraints for rice production. A study was carried out to analyze the virulence, genetic diversity, and population structure of Xoo isolates collected from different rice ecosystems of Karnataka, India. The taxonomic identity of all 54 isolates was confirmed using a Xoo-specific PCR assay. The virulence assay on a set of twenty-eight near-isogenic differentials identified nine pathotype groups (KPthX-1 to KPthX-9). Among them, KPthX-6 was highly virulent, whereas KPthx-2 was less virulent. Differentials with four and five resistance (R) genes (Xa4+xa5+xa13+Xa21, Xa4+Xa7+xa13+Xa21, and Xa4+xa5+Xa7+xa13+Xa21) were effective against all the pathotypes. Further, genetic diversity was deduced using a universal marker set, JEL-1, and JEL-2, complementary to a conserved repetitive sequence IS1112. The amplicon size and numbers varied from 100 bp to 3 Kb and 1 to 25. Based on the amplicon counts, Jaccard’s co-efficient and phylogenetic analyses were carried out and categorized the 54 isolates into three clusters (I to III). Cluster-II contains the maximum number of isolates (27), followed by cluster-I (23 isolates) and cluster-III (one isolate). The structure analysis categorized the isolates into five subgroups, viz. SG1, SG2, SG3, SG4, and SG5 indicate the existence of five subpopulations. The present study has identified the genetic and virulence diversity of the Xoo population and reported the effective R gene/s for different rice ecosystems, which can be used in the breeding program for the eco-specific management of BLB of rice.