The seven selected primers OPA-02, OPA-04, OPA-18, OPD-07, OPD-08, OPD-15 and OPD-16 were used to generate unique shared loci to each species and shared loci by the two species. The hierarchical dendrogram indicates three main branches: cluster 1 (RORETZI 01~RORETZI 11) and cluster 2 (HILGENDORF 12~HILGENDORF 22) from two geographic populations of ascidians, Halocynthia roretzi and H. hilgendorfi. The shortest genetic distance displaying significant molecular difference was between individuals' HILGENDORF no. 14~HILGENDORF no. 19 (genetic distance =0.008). Ultimately, individual no. 02 of the RORETZI ascidian was most distantly related to HILGENDORF no. 21 (genetic distance=0.781). These results demonstrate that the H. roretzi population is genetically different from the H. hilgendorfi population. From what has been said above, the potential of PCR analysis to identify diagnostic markers for the identification of two ascidian populations has been demonstrated. Generally speaking, using a variety of decamer primers, this PCR method has been applied to identify specific markers particular to line, species and geographical population, as well as genetic diversity/polymorphism in diverse species of organisms.