Abstract: The internal transcribed spacer 2 (ITS 2) and the flanked genes fragment in pearl oyster Pinctada fucata was studied, including samples collected from three geographic localities: China,Japan and Australia. The PCR product contains 5.8 S gene, partial sequence, ITS 2, complete sequence and 28 S gene, partial sequence. Total 16 genotypic sequences were analyzed. Sequences of 5.8 S and 28 S segments are highly conservative and are 64 and 249 bp long, respectively with exclusion of primers base pairs. The length of ITS 2 varies from 230 to 237 bp with twenty mutation sites,including twelve insertion/deletion sites, four singletons and four parsimony informative sites, showing highly genetic variation. The GC contents of 5.8 S (59.4%) and 28 S (58.1%) segments are higher than AT contents, and also higher than that of ITS 2 (51.3%～52.0%). Base frequency of T is the lowest in 28 S segment and the base frequency of A the lowest in 5.8 S. The genetic distances within and among the three populations are low (0.010～0.013) and overlapped. The genotypic data reveals that one genotype is shared by three populations and one shared by Japanese and Australian populations; most genotypes are population specific. The sequence information indicates that there are no common mutation sites for individual population. These findings suggest that the three populations may diverge at recent and/or there are gene flows among them.Thus selective breeding may profit from the high level of genetic variation in the populations of P. fucata.