In this paper we analysed the RFLP of mtDNA in Lanzhou (LZ) population of D. virilis. By reanalysing the RFLP data of our previous work on other natural populations of D. virilis, a phylogenetic tree was produced based on the UPGMA method. It shows three main clusters: the Northern populations (LZ QD), the East China populations (NJ, SH, NB) and the Southern population (QZ). With the mtDNA's RFLP data and the results of our former work on allozyme variation in natural populations of D. virilis, we suggest that there exists a latitudinal cline of genetic variation in natural populations of D.virilis. The mechanism for the maintenance of the observed latitudinal pattern is discussed.