Wheat hybrid necrosis refers to the abnormal necrosis of leaves in hybrid progenies, which is caused by the interaction of two dominant complementary genes Ne1 and Ne2 present separately in some normal parents. Necrosis phenotype severely reduced the yield and quality of wheat and was regarded as a barrier to gene transfer in wheat breeding. In order to fine mapping the two Ne genes, BSR-Seq and SLAF-Seq were performed using sample pools consisted of common wheat Zhoumai 22 and N9134, and their derived F1, F2 and BC1F1 populations. Based on the SNP information obtained by the two methods of sequencing, we developed KASP and AS-PCR markers. Using the corresponding BC1F1 populations of Zhoumai 22 and N9134, Ne1 was fine mapped on chromosome 5B, while Ne2 was on chromosome 2B. In order to determine the distribution of the two Ne genes in various wheat regions in China, we screened the genotypes of 1364 wheat varieties (lines) by hybridization and closely linked molecular markers. We concluded that 26.2% and 33.2% of the 1,364 cultivars (lines) were genotyped with Ne1Ne1ne2ne2 and ne1ne1Ne2Ne2, respectively. And our results demonstrated discrete differences in the distribution and proportion of Ne genes from different wheat region, with highly variable correlation coefficients among the regions.