The sequences of the mitochondrial ND4-ND4L were determined for all the 33 extant taxaes of the Drosophila melanogaster species group, so evolutionary traits of ND4 gene sequences were analyzed. We found a strong transition bias in closed related species and saturation of transitional substitutions in distant related species. Moreover, we calculated the diverge time of eight subgroup according to the Tamura-Nei distance between D. melanogaster and D. yakuba. Our result suggested that ananassae subgroup branched off firstly, and montium, melanogaster, ficsphila, eugracilis, elegans, suzukii-takahashii branched off sequentially.