Qfhi.nau-4B is a major quantitative trait locus (QTL) against Fusarium graminearum infection identified in the Fusarium head blight-resistant germplasm Wangshuibai. To fine map this QTL, a recombinant inbred line (RIL) population of 530 lines derived from Nanda2419 x Wangshuibai and the BC(3)F(2) population derived from the cross of a Qfhi.nau-4B near isogenic line (NIL) with susceptible cultivar Mianyang 99-323 as the recurrent parent were screened for recombinants occurred between microsatellite markers Xbarc20 and Xwmc349 that flank Qfhi.nau-4B. A total of 95 recombinants were obtained, including 45 RIL recombinants obtained through reverse-selection of Qfhi.nau-5A and 50 NIL recombinants from the BC(3)F(2) population. Genotyping these recombinant lines with 22 markers mapping to the Xbarc20 and Xwmc349 interval revealed fourteen genotypes of the RIL recombinants as well as of the NIL recombinants. Two-year field evaluation of their resistance to Fusarium infection showed that these lines could be clearly classified into two groups according to percentage of infected spikes. The more resistant class had over 60% less infection than the susceptible class and were common to have Wangshuibai chromatin in the 1.7-cM interval flanked by Xhbg226 and Xgwm149. None of the susceptible recombinants had this Wangshuibai chromatin. Qfhi.nau-4B was thus confined between Xhbg226 and Xgwm149 and named Fhb4. The interval harboring Fhb4 was mapped to 4BL5-0.86-1.00 bin using Chinese Spring deletion lines, a region with about 5.7 times higher recombination rate than the genome average. This study established the basis for map-based cloning of Fhb4.