Genetic status of Acanthamoeba spp. were tested on the basis of random amplified polymorphic DNA (RAPD) marker analysis. Four previously established Acanthamoeba species, 4 Korean isolates of Acanthamoeba sp., and one American isolate of Acanthamoeba sp. were analyzed by RAPD-PCR using an arbitrary decamer primers. Amplification products were fractionated by agarose gel electrophoresis and stained by ethidium bromide. Eighteen primers produced DNA amplification profiles revealing clear differences among 4 species. Nine of them also produced DNA amplification profiles which included some isolate-specific amplification products. On the basis of amplified fragments by 18 primers, the pairwise similarity indices between A. culbertsoni and other species (i.e., A. hatchetti, A. triangularis, A. polyphaga) were 0.300, 0.308, and 0.313, respectively. Similarity index between A. hatchetti and A. triangularis was 0.833. The mean similarity index among the 3 Korean isolates (YM-2, -3, -4) was 0.959 and 0.832 among them and 2 other species (A. hatchetti and A. triangularis). The mean similarity index among YM-5 and other Korean isolates (YM-2, -3, -4) was 0.237. However, the similarity index between YM-5 and A. culbertsoni was 0.857, which suggests that YM-5 is genetically more similar to A. culbertsoni than other Korean isolates. Phenogram reconstructed by UPGMA method revealed that there are two groups: one group consists of A. hatchetti, A. triangularis, and 3 Korean isolates (YM-2, -3, -4), and the other group consists of A. culbertsoni, A. polyphaga, HOV, and YM-5.