The fragments of CO Ⅰgene of 13 populations of Dugesia japonica collected from Yunnan Province, China were amplified via PCR, and then cloned and sequenced. The sequences were aligned and analyzed with Clustal X 1.80 and MEGA 4.0, and the maximum parisimony (MP) tree and Bayes tree were reconstructed via PAUP^*4.0 and MrBayes 3.1.1, respectively. The results show that the A+T contents of 13 sequences have little difference, ranging from 67.1% to 68.8%, more than the G+C contents obviously. The pairwise genetic distances based on model Kimura 2-parameter between sequences range from 0 to 0.199.Distances among populations of Yuxi, Dali2 and Lincang1 were 0, that between Puer and Xishuang was the largest (0.199). The MP tree and the Bayes tree almost show the same topology. Based on the clustering pattern of partial CO Ⅰ genes and the geographical features of Yunnan, it is hypothesized that the freshwater planarian D.japonica in Yunnan Province could have originated from the central sites, then diffused to the marginal sites, or vice versa. More data are needed to test the hypothesis.