Rice, a staple crop and lifeline for millions, is India’s second most important crop after wheat, with the country being one of Asia’s leading producers. However, bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) poses a severe threat to rice production across both tropical and temperate regions due to its high epidemic potential. This study focused on isolating and evaluating the virulence of various Xoo isolates from different rice-growing areas in Tamil Nadu under controlled pot trials. Among the isolates, Xoo3 was identified as the most virulent, causing a BLB severity of 43.9%, while Xoo6 showed the lowest virulence with a 29.8% severity rate. These findings highlight the variability in pathogenicity among Xoo isolates and underscore the importance of targeted disease management practices to mitigate BLB impact on rice yields in affected regions.