The relationships within Drosophila melanogaster species group are controversial from morphology, chromosomes and DNA sequences. This study utilises a molecular approach aimed at uncovering the phylogenetic relationships among 33 taxa representing 8 subgroups of D. melanogaster species groups. Mitocondrial ND4L-ND4 was sequenced in the all 8 subgroups covering a wide geographic area. MP and Bayesian analysis produced an identical tree topology with relatively strong support in most nodes. It reveals that the melanogaster species group clustered in three main lineages：1）montium subgroup; 2） ananassae subgroup; 3） Oriental subgroups （ melanogaster, ficsphila, eugracilis, elegans, syzukii and takahashii）. The montium subgroup branched off first, followed by the ananassae subgroup. In the third lineage, melanogaster is the most divergent subgroup followed by ficsphila, eugracilis, elegans in that order. The suzukii and takahashii sister subgroups are the last to branch off.
