Abstract A few EST-derived STS markers localized on Qfhs.ndsu-3BS, a major QTL for resistance to Fusarium head blight (FHB) in wheat, have been previously identified in the ‘Sumai 3’/‘Stoa’ population. In this study, we used a ‘Wangshuibai’ (resistant) /‘Seri82’ (susceptible) derived population, linkage group, QTL, and quantitative gene expression analysis to assess the genetic background dependence and stability of the EST-derived STS markers for use in marker aided selection to improve FHB resistance in wheat. Based on our results, a QTL in the map interval of Xsts3B-138_1− Xgwm493 on chromosome 3BS was detected for FHB resistance, which accounted for up to 16% of the phenotypic variation. BLASTN analysis indicated that Xsts3B-138_1 sequence had significant similarity with the resistance gene analogue. Real-time quantitative PCR showed that the relative expression of Xsts3B-138_1 in ‘Wangshuibai’ at 96 h after inoculation was 2.6 times higher than ‘Seri82’. Our results underlined that EST-derived STS3B- 138 markers could be predominantly used in marker aided selection to improve FHB resistance in wheat.