The importance of genetic resources lies in their pivotal role in improving the quality of crop varieties, particularly in enhancing their resilience to both biological and environmental stresses. One such challenge is Spot blotch (SB), a destructive leaf disease that primarily affects wheat, especially in warm and humid regions, particularly the eastern parts of South Asia, including the eastern Gangetic plains and the Peninsular zone. Developing wheat cultivars with resistance to SB stands out as the most effective strategy for managing this disease. Therefore, the primary objective of the current study was to validate the resistance to SB in one hundred twenty-seven, dicoccum wheat germplasm accessions. This validation process relied on both observable traits and genetic characteristics. The germplasm accessions were obtained from various sources, including the International Center for Agricultural Research in the Dry Areas in Lebanon, the International Maize and Wheat Improvement Center in Mexico, and the International Maize and Wheat Improvement Center (CIMMYT) in Mexico. The evaluation for Spot blotch resistance was conducted in the field under controlled conditions of disease prevalence during the years 2020–21 and 2021–22. The assessment involved two vulnerable control cultivars (DDK-1025 and Sonalika) and two confirmed resistant control cultivars (Chirya-3 and HI-8663). Out of the 127 germplasm accessions tested, forty displayed resistance to SB, confirmed through a specific genetic marker known as Xgwm120. Notably, four accessions – Acc. GPM Dicoccom-IR-76, Acc. GPM Dicoccom-IR-98, Acc. GPM Dicoccom-IR-102, and ICARDA-14-127687 – exhibited even greater resistance than the well-established Spot blotch-resistant genotype, Chirya-3. These promising lines with resistance potential offer significant prospects for wheat breeders dedicated to developing spot blotch resistant wheat lines.