The parasitoid Diachasmimorpha longicaudata complex in Thailand contains at least 3 cryptic species informally designated as species D. longicaudata A, B and BB. DNA sequence data of nuclear ITS2 (second internal transcribed spacer) were used to characterize members of this D. longicaudata complex. The polymerase chain reaction (PCR) amplicon of ITS2 region of D. longicaudata B ($$sim_sim$$ 650 bp) clearly differentiated this species from A and BB (amplicon of $$sim_sim$$ 590 bp). Sequence alignment of individual parasitoids revealed that low intraspecies differences ranged from 0.457 to 3.991%, but interspecies differences ranged from 7.566 to 12.989%. Phylogenetic trees constructed using Neighbor-Joining (NJ) and Maximum Parsimony (MP) methods, taking the parasitoid Psyttalia concolor complex as an outgroup, revealed that D. longicaudata A, B and BB formed a monophyletic group, with species A and BB being more closely related than species B. ITS2 characterization of D. longicaudata complex has revealed an interesting divergence of the three cryptic sibling species in Thailand.