This study identified Phalacronotus to species level and examined its morphometric variation among three collection localities, and between sexes, in the Mekong Delta, Vietnam. Specimens were collected from local markets and fishers, including An Giang (n = 36) and Can Tho (n = 36) in the Hau River and Tien Giang (n = 30) in the Tien River for morphological measurements and DNA barcoding analysis. Eight mitochondrial cytochrome oxidase subunit I (COI) sequences of Phalacronotus sp. shared 99.6 ± 0.23 % similarity with five GenBank (GB) P. bleekeri sequences and differed by 7.6 % to 13.3 % from seven GB P. apogon sequences. The Vietnamese specimens of Phalacronotus were similar to P. bleekeri in number of anal fin rays (75–80) and the shape of the upper jaw teeth. Thus, the COI sequences and these morphological characteristics indicate that Phalacronotus sp. samples collected in the Mekong Delta are Phalacronotus bleekeri (Günther, 1864). Morphometric characteristics differed between populations in 15 of 19 indices (P < 0.05) and between females and males in three characteristics relating to the belly. Head characteristics, body depth, and caudal peduncle depth were the main parameters contributing to the inter-population variation. Based on size-adjusted data, 96.7 % of specimens were correctly classified from the Tien Giang population, while 27.8 % and 33.3 % were misclassified between the Can Tho and An Giang populations, respectively. These results indicate that morphometric parameters of P. bleekeri varied mainly between populations in the two Mekong River tributaries.