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Fusarium head blight (FHB) of wheat poses a serious threat to food security in the Yellow-Huai River Valley Winter Wheat Region (YHW) of China. Discovery of new resistant quantitative trait loci (QTLs) or genes and application of them to highly susceptible varieties in the YHW are of great significance for ensuring the grain yield. Here, 160 recombinant inbred lines (RILs) from the cross between N553 (resistant) and Yangmai 13 (moderately susceptible) were used to evaluate FHB resistance by point inoculation, spray inoculation, and natural infection. A high-density genetic map was constructed by using a 15K SNP array and 128 polymorphism SSR markers. A total of 1452 polymorphic markers were identified, which formed 21 linkage groups and covered a total of 3555.1 cM in length. Two and four QTLs respectively related to type I and type II resistance were detected, among which QFhb-hnau.3BS.1 and QFhb-hnau.2DL were stably identified in most environments in Yangzhou and Zhengzhou, whereas QFhbn-hnau.5AL was only identified under natural infection in Jianyang. Based on the physical position (IWGSC RefSeq v1.0), QFhb-hnau.3BS.1 from the landrace N553 is likely to be Fhb1, while QFhb-hnau.2DL from Yangmai 13 may be a novel QTL. Significantly higher FHB resistance was observed in the lines with both QFhb-hnau.3BS.1 and QFhb-hnau.2DL, indicating that these two QTLs have apparent additive effects, and the RILs harboring both the two QTLs may have great application potential for the improvement of FHB resistance in wheat breeding. [ABSTRACT FROM AUTHOR]