A 327 bp region of the mitochondrial DNA NADH dehydrogenase 4 subunit (ND4) was analyzed for genetic variation in 43 wheat midges (Sitodiplosis mosellana) from 12 different areas of China. Nucleotide sequencing revealed 21 polymorphic sites and 22 distinct haplotypes. Haplotypes was closely related with geographical distribution based on Neighbor-Joining analysis. The haplotypes from the spring wheat region samples clustered into one group, and the majority of winter wheat region samples clustered into another group. However, the haplotypes in the Chang'an population in Shaanxi Province (SC) appeared synchronously in each group. Thus, the distribution area of the SC population became a transition place of haplotype distribution. The genetic distances among geographic populations of the wheat midges were 0.0000∼0.0180 and those within each populations were 0.0000∼0.0145.