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Background The Vietnamese Mekong Delta (VMD) is the granary for the whole country, providing animal and plant resources, especially fish. Among the fish species, the genus Glossogobius are the majority. Until now, research for this species has been solely relied on fish morphology for identification. Hence, the present study aimed to describe the morphological variations of the morphologically identified gobies and to validate them at the molecular level through the sequencing of the barcode region, the mitochondrial cytochrome C oxidase subunit I (COI) gene to preliminary provide fundamental information for conservation. Results The mitochondrial cytochrome C oxidase subunit I genes were amplified successfully with an approximate size of 650-680 bp. Their morphometries were quite different, and the genetic distance (p-value) among groups and within groups ranged from 0.00 to 0.12. The similarity of the COI gene sequences between the analyzed samples and in the NCBI database was from 87.01 to 100%. The specimens of G. aureus, G. giuris and G. sparsipapillus were interspersed in small branches of the phylogenetic tree with a low genetic distance highlighting that the genetic diversity of COI gene was low among species. Therefore, it is recommended that a combination of morphological method and mtCOI DNA barcoding is required for accurate classification. Conclusion This study helps determine three distinct lineages of Glossogobius species, so an appropriate strategy can be proposed for exploitation and conservation.