Simple SummaryThis study evaluated 26 sequences of terrapins worldwide through COI DNA barcoding and phylogenetic analysis, which included 12 species and three families. Moreover, 16 haplotypes were found; they were either misidentified, or a potential cryptic species was determined betweenB. baskaandB. affinis affinis. Thus, COI remains an effective barcode marker for the terrapin species.AbstractTechnological and analytical advances to study evolutionary biology, ecology, and conservation of the Southern River Terrapin (Batagur affinisssp.) are realised through molecular approaches, including DNA barcoding. We evaluated the use of COI DNA barcodes in Malaysia’s Southern River Terrapin population to better understand the species’ genetic divergence and other genetic characteristics. We evaluated 26 sequences, including four from field specimens of Southern River Terrapins obtained in Bota Kanan, Perak, Malaysia, and Kuala Berang, Terengganu, Malaysia, as well as 22 sequences from global terrapins previously included in the Barcode of Life Database (BOLD) Systems and GenBank. The species are divided into three families: eight Geoemydidae species (18%), three Emydidae species (6%), and one Pelomedusidae species (2%). The IUCN Red List assigned the 12 species of terrapins sampled for this study to the classifications of critically endangered (CR) for 25% of the samples and endangered (EN) for 8% of the samples. With new haplotypes from the world’s terrapins, 16 haplotypes were found. The intraspecific distance values between the COI gene sequences were calculated using the K2P model, which indicated a potential cryptic species between the Northern River Terrapin (Batagur baska) and Southern River Terrapin (Batagur affinis affinis). The Bayesian analysis of the phylogenetic tree also showed both species in the same lineage. The BLASTn search resulted in 100% of the same species ofB. affinisasB. baska. The Jalview alignment visualised almost identical sequences between both species. The Southern River Terrapin (B. affinis affinis) from the west coast of Peninsular Malaysia was found to share the same haplotype (Hap_1) as the Northern River Terrapin from India. However,B. affinis edwardmollifrom the east coast of Peninsular Malaysia formed Hap_16. The COI analysis found new haplotypes and showed that DNA barcodes are an excellent way to measure the diversity of a population.