Rice false smut, caused by the fungus Ustilaginoidea virens is one of the most destructive and re-emerging disease in many rice growing areas of Tamil Nadu, India. The current study was carried out to characterize different isolates of Ustilaginoidea virens collected from major rice growing areas in Tamil Nadu, India. The pathogen was isolated by different isolation methods, out of which inner spore dust was effective. The colony was milky white to yellowish white, fluffy, leathery and compact. The colony diameter ranged between 35.33 mm and 65.33mm. The mycelium was hyaline, septate and width of the hyphae ranged between 1.84 and 3.10 µm. The size of the conidia was also varied among the isolates and the maximum size (6.35 µm) was observed in FS 6 isolate and minimum in FS 2 (4.10 µm). The growth of fungus in different media was examined. The maximum colony diameter was observed in Potato Sucrose Agar (PSA) medium and minimum growth in Czapek Agar (CA) medium. The molecular confirmation was done through U. virens-specific internal transcribed spacer (ITS) region amplification using the primers, US 1-5/US3-3. The primers amplified a product of 380 bp which are specific for the fungus U. virens. The identity of the fungus was further confirmed through ITS sequencing which showed 98-99% sequence homology. The dendrogram showed two main clusters; cluster I comprised of five isolates (FS1, FS2, FS9, FS10 and FS3), while Cluster II consisted of five isolates (FS4, FS8, FS6, FS5 and FS7).