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The mangrove horseshoe crab, Carcinoscorpius rotundicauda, has divergent populations between the east and west coasts of Peninsular Malaysia, with the southern coast acting as a land barrier. The actual position of such a genetic break along Peninsular Malaysia as well as the connectivity status of the southernmost C. rotundicauda populations with east and west coast populations remain unexplored, however.The aim was to investigate the genetic diversity and structure of C. rotundicauda populations from the west (Kuala Sepetang in State Perak), east (Balok in State Pahang), and southern (Pendas in State Johor) coasts of Peninsular Malaysia. Haemolymph samples from adult C. rotundicauda specimens (n = 152) and eggs from their freshly deposited nests (n = 190) were collected monthly (from January 2016 to January 2017) for the sequencing of mitochondrial cytochrome c oxidase subunit I (COI).Gene isolates of C. rotundicauda from the present study were compared with National Center for Biotechnology Information (NCBI) GenBank sequences to cover most of the range of the species in Asia. A neighbour‐joining tree strongly supported two clades, separating the west‐coast populations from the south‐ and east‐coast populations, with further substructure patterns.Both haplotype network and barrier analyses revealed a genetic break within the Strait of Malacca instead of the southern tip of Peninsular Malaysia. The southernmost samples from the Strait of Johor formed a haplotypic diverse gene pool that appeared only as a subclade of the eastern populations. In a detailed haplotype network of 347 individuals, individuals with similar COI sequences indicate connectivity between C. rotundicauda on the east and C. rotundicauda on south and south west of Peninsular Malaysia.Overall, the genetic break between C. rotundicauda populations is better explained by the convergent ocean currents and available mangrove habitats on the west coast (i.e. Strait of Malacca), rather than the point of the Malay Peninsula acting as a land barrier alone. [ABSTRACT FROM AUTHOR]