One hundred and twenty isolates of Bipolaris sorokiniana of wheat were isolated, identified and characterized based on morphological, cultural, pathogenic and ITS (Internal Transcribed Spacer) based molecular parameters. Collected isolates differed in respect of radial mycelial growth rate, color of the colony, surface texture of the colony, shape of the colony, conidia production ability and shape and color of conidia. ITS1 and ITS4 based PCR products yielded a species-specific band of 600 bp (base pairs) on agarose gel for the confirmation of B. sorokiniana. The mycelial growth rate of the isolates ranged from 1.39 to 4.46 mm/day. The septation of conidia ranged from 1.4 to 8.6. On the basis of conidia morphology, the isolates were grouped into five different groups, whereas the isolates were grouped into 12 cultural groups based on cultural characteristics. Cultural group 8 [CG (Cultural Group)-8] was the most virulent [38.5% LAD (Leaf area diseased)] and [CG (cultural Group)-4] was the less virulent [2% LAD (Leaf area diseased)] group among 12 groups when inoculated at tillering stage of wheat cv. Kachan.