Genetic data are essential for clarifying species boundaries and informing conservation of mudskippers, yet molecular studies on Periophthalmus species in the Mekong Delta remain limited. This study provides the first mtCOI-based assessment of Periophthalmus gracilis and Periophthalmus variabilis across four coastal provinces of Vietnam. A total of 24 sequences were generated and analyzed using phylogenetic approaches. Both species were resolved into well-supported monophyletic clades in both Maximum Likelihood and Bayesian analyses, with high interspecific divergence and low to moderate intraspecific variation. Genetic differentiation was observed primarily between species, whereas within-species variation among localities appeared weak in this limited mtCOI dataset. Two P. variabilis individuals from Soc Trang formed a separate lineage, which should be interpreted cautiously due to limited sampling and reliance on a single mitochondrial marker. These results expand upon previous Cytb-based work and establish a preliminary mtCOI baseline for Periophthalmus in the Mekong Delta. Accurate species delimitation is critical for biodiversity assessment, habitat management, and conservation of mangrove ecosystems, and future studies integrating nuclear markers and broader sampling will be necessary to test for cryptic diversity and assess connectivity.

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