The Glossogobius species play an essential role in food supply and are distributed widely from the marine to freshwater, especially in the Mekong Delta, Vietnam (VMD). Some of their morphometrics and meristics are observed to vary with species and sampling sites. Therefore, the present study aims to verify if their mitochondrial Cytochrome b (Cytb) gene, one of the popular gene sequences used in fish phylogenic variation assessment, varies with species and sampling sites in the VMD. The Cytb gene size was 1300 bp for GcytbH/GcytbL primer pair and 1045 bp for GluMuq1-F/Mixcyto937-2R. The genetic distances within and among these three fish species groups were 0–11%. The Cytb gene sequences’ similarity between this study and the NCBI database was 85.84–100%. The Glossogobius specimens were observed to disperse in small branches of the phylogenetic tree with a low K2P value, suggesting that the Cytb genetic diversity may be low among species.