Spot blotch, incited by Bipolaris sorokiniana, is one of the serious foliar diseases affecting wheat productivity worldwide, significantly. The cultural, morphological, pathogenic, and molecular variability were studied comprehensively among 51 B. sorokiniana isolates collected from diverse wheat-growing regions of India. Regarding cultural variability, the highest radial growth rate was observed in isolate Bs-9 (8.45 mm/day), whereas isolate Bs-27 exhibited the lowest growth rate (2.50 mm/day). Morphological analysis indicated that isolate Bs-3 had the greatest conidial length, conidial breadth, and number of septa, whereas isolate Bs-12 displayed the minimum values for these traits. A highly significant positive correlation was observed among conidial length, conidial breadth, and number of septa. Pathogenic variability analysis identified isolate Bs-3 (Manikchak, West Bengal) as the most virulent, exhibiting the shortest incubation period (2.75 days), the largest mean lesion length (5.05 mm) and lesion breadth (1.69 mm), maximum lesion size (8.53 mm2), lesion coverage (67.50%), and the highest disease score (4.5). A highly significant negative correlation (r = −0.87**, p < 0.001) was observed between incubation period and disease score. Population structure analysis using 28 SSR markers separated the isolates into two distinct sub-populations (SP1 and SP2). Analysis of molecular variance (AMOVA) indicated that 71% of the total variation was due to differences within subpopulations, whereas 29% was attributed to variation between subpopulations. The mean number of effective alleles (Ne) was 7.118 for SP1 and 8.333 for SP2. Shannon’s information index (I) was higher in SP2 (2.238) than in SP1 (2.011). SP2 also exhibited greater genetic diversity, as reflected by higher expected heterozygosity (He = 0.880) and unbiased heterozygosity (uH = 0.917) compared to SP1. The results demonstrated significant variation among B. sorokiniana isolates in morphological, cultural, pathogenic, and molecular attributes. Notably, isolate Bs-3 was identified as a highly virulent strain and may serve as a reference isolate for rigorous screening of wheat germplasm and for detailed studies of wheat- B. sorokiniana interactions.