Hessian fly [Mayetiola destructor (Say)] is one of the most destructive insects in common wheat (Triticum aestivum L.). Host resistance is considered the most effective means of control. ‘Pioneer 26R61’ showed good resistance to Hessian fly biotype E at low temperature (16°C), and partial resistance at high temperature (24°C). ‘AGS 2000’ was susceptible at both temperatures. Mapping population (178 F6:7 RILs) from a cross between Pioneer 26R61 and AGS 2000 were evaluated to the Hessian fly biotype E at low temperature. Whole genome survey revealed a novel major QTL QHf.uga-6AL responsible for the seedling resistance in Pioneer 26R61, and it was flanked by DArT markers wPt-731936 and wPt-732975, explaining nearly 50% of total phenotypic variation. QHf.uga-6AL was also identified when the population was infested with the Hessian fly biotype vH13, which was virulent to H13, and contributed 23% of the phenotypic variation (LOD, 9.6). Another QTL, QHf.uga-3DL (R2, 7%; LOD, 3.9), was also detected on 3DL with resistance to vH13, showing close linkage with SSR marker Xgdm8. When evaluated at later stage that some lines apparently recovered from Hessian fly damage, the R2 and LOD value of QHf.uga-6AL became lower (R2, 14%; LOD, 6.4), whereas QHf.uga-3DL was higher (R2, 11%; LOD, 5.1). In summary, QHf.uga-6AL conditioned seedling resistance for both Hessian fly biotypes and QHf.uga-3DL for adult plant resistance to biotype vH13. Marker data also indicated QHf.uga-3DL was different from H24, H26 and H32 on chromosome 3DL.