Spot blotch (SB) is an important fungal disease of wheat in South Asia and South America. Host resistance is regarded as an economical and environmentally friendly approach of controlling SB, and the inheritance of resistance is mostly quantitative. In order to gain a better understanding on the SB resistance mechanism in CIMMYT germplasm, two bi-parental mapping populations were generated, both comprising 232 F2:7 progenies. Elite CIMMYT breeding lines, BARTAI and WUYA, were used as resistant parents, whereas CIANO T79 was used as susceptible parent in both populations. The two populations were evaluated for field SB resistance at CIMMYT’s Agua Fria station for three consecutive years, from the 2012–2013 to 2014–2015 cropping seasons. Phenological traits like plant height (PH) and days to heading (DH) were also determined. Genotyping was performed using the DArTSeq genotyping-by-sequencing (GBS) platform, and a few D-genome specific SNPs and those for phenological traits were integrated for analysis. The most prominent quantitative trait locus (QTL) in both populations was found on chromosome 5AL at the Vrn-A1 locus, explaining phenotypic variations of 7–27%. Minor QTL were found on chromosomes 1B, 3A, 3B, 4B, 4D, 5B and 6D in BARTAI and on chromosomes 1B, 2A, 2D and 4B in WUYA, whereas minor QTL contributed by CIANO T79 were identified on chromosome 1B, 1D, 3A, 4B and 7A. In summary, resistance to SB in the two mapping populations was controlled by multiple minor QTL, with strong influence from Vrn-A1.