We examined Daphnia pulex in Japan to clarify if they were representative of indigenous populations or colonized recently. Phylogenetic analysis of mtDNA suggests that Japanese lineages of D. pulex are immigrants from North America, and are hybrids formed with Daphnia pulicaria prior to this immigration. Based on the mtDNA sequences, the D. pulex individuals aggregated into four distinct genetic groups (JPN 1‐4) comprising a total of 21 haplotypes. Surprisingly, microsatellite analysis with 12 loci revealed only a single multilocus genotype per genetic group, suggesting that D. pulex populations in Japan are comprised of asexual individuals, derived from only four clones. According to the reported mutation rate of mtDNA, JPN 1 and 2, now widely distributed across Japan, were estimated to have invaded between 680 yr and 3400 yr ago, while JPN 3 and 4 colonized much more recently. Results also indicated that the invasion of some clones could not be attributed to recent human activities and most likely occurred by rare natural events. Since the evolutional longevity of asexual clones is thought to be limited, genetic diversity of D. pulex in Japan has the possibility of decreasing in the near future without addition of novel gene flow.