Genetic resources are of paramount importance for developing improved crop varieties, particularly for biotic and abiotic stress tolerance. Spot blotch (SB) is a destructive foliar disease of wheat prevalent in warm and humid regions of the world, especially in the eastern parts of South Asia. For the management of this disease, the most effective measure is the development of resistant cultivars. Thus, the present investigation was carried out to confirm SB resistance in 200 germplasm accessions based on phenotypic observations and molecular characterization. These elite breeding lines obtained from the International Centre for Maize and Wheat Improvement, Mexico, are developed deploying multiple parentages. These lines were screened for SB resistance in the field under artificially created epiphytotic conditions during 2014–15 and 2015–16 along with two susceptible checks (CIANO T79 and Sonalika) and two resistant checks (Chirya 3 and Francolin). Eighty-two out of 200 germplasm accessions were found resistant to SB and resistance in these lines was confirmed with a specific SSR marker Xgwm148. Three accessions, VORONA/CNO79, KAUZ*3//DOVE/BUC and JUP/BJY//URES/3/HD2206/HORK//BUC/BUL were observed possessing better resistance than the well-known SB-resistant genotype Chirya3. These newly identified resistant lines could be used by wheat breeders for developing SB-resistant wheat varieties.