Grain spot disease in rice, caused by Curvularia lunata, poses a significant threat to rice production, leading to substantial yield losses. Aim of the study is to isolate, identify, and evaluate the pathogenicity of C. lunata from infected rice grains in Tamil Nadu, India. The pathogen was isolated using single hyphal tip and spore isolation techniques, followed by morphological and molecular identification. Colonies exhibited rapid growth on Potato Dextrose Agar (PDA), with distinct greyish-brown pigmentation. Light and scanning electron microscopy confirmed the characteristic conidial morphology of C. lunata. Molecular identification using ITS region sequencing confirmed the species identity, with the isolate showing 89% genetic homology to previously reported C. lunata strains. Pathogenicity tests confirmed the fungus as the causal organism of grain spot disease, as inoculated plants developed characteristic lesions, fulfilling Koch’s postulates. Growth analysis on different media revealed PDA as the most suitable for optimal mycelial development and sporulation. The findings emphasize the necessity of early detection and effective management strategies to mitigate C. lunata-induced grain spot disease in rice. Future research should focus on the genetic diversity of C. lunata isolates, host-pathogen interactions, and resistance breeding in rice. Additionally, exploring eco-friendly biocontrol agents and integrated disease management approaches could offer sustainable solutions. This research contributes to the understanding of C. lunata as an important rice pathogen and underscores the need for continued surveillance to prevent potential outbreaks in rice-growing regions worldwide.