In this article, RAPD was employed to detect the DNA polymorphism of Carchesium polypinum in three lakes: Nanhu Lake (N), Shahu Lake (S) and Donghu Lake (D). The results were used to deduce the definition of the populations: (1) The 20 primers produced 172 clear bands with an average of 8.6 fragments ranging from 100bp to 1600bp. The other 11 primers produced 59 legible bands with an average of 5.4 fragments ranging from 100bp to 1 500bp; (2) SPSS 12.0 and RAPDistance 1.04 that had similar trees were composed of two parallel main branches. Samples from Shijizhong (Z) of D, Shuiguohu (H) of D and N clustered into a branch and another sample from D — Fengguangcun (F) — gathered with S or made another main branch itself. The result that the three samples from D were separated by the samples from other lakes suggested that the individuals of C. polypinum in D belong to several populations. This discovery will promote micro-hydrobiont population ecology research. In addition, it will help in the study of the essence of the transitional zone and its dispersal and vicariance behavior. We believe that this study will contribute to the study of population differentiation and the mechanism of speciation.