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Background Bigfin squid is one of the economically important seafood resources in Vietnam's fisheries and the waters around Con Dao and Phu Quoc islands are two major fishing grounds where this species has been actively exploited. The start codon targeted polymorphism (SCoT) and CAAT box--derived polymorphism (CBDP) techniques were used to generate DNA fingerprinting data to analyze the genetic diversity, variation, and structure of the two populations in the waters surrounding Phu Quoc and Con Dao islands together with mitochondrial cytochrome C oxidase subunit I (COI) gene sequence data. Results Con Dao population possessed a higher diversity [expected heterozygosity (He) = 0.2254, Shannon index (I) = 0.3459, percentage of polymorphic bands (PPB) = 80.14%, nucleotide diversity (π) = 0.0336, haplotype diversity (h) = 0.910 with 16 haplotypes] than Phu Quoc population (He = 0.1854, I = 0.2873, PPB = 70.38%, π = 0.0246, h = 0.838 with 14 haplotypes). The genetic diversity at species level in the investigated region was at level of He = 0.2169, I = 0.3399, PPB = 86.41, π = 0.0289, and h = 0.892 with 24 haplotypes. Based on DNA fingerprinting data, the pairwise genetic similarity coefficients among individuals of the Con Dao population were lower (average of 0.7977) than the Phu Quoc population (average of 0.8316). Based on mitochondrial COI data, the pairwise genetic distances among individuals of the Con Dao population were higher (average of 0.0361) than the Phu Quoc population (average of 0.0263). Gene differentiation (GST) between two investigated populations was 0.0316 and 0.0310 leading to the genetic distance was 0.0573 and 0.0213 and the gene flow between them was Nm = 8.2209 and 11.4700 migrants per generation among populations based on DNA fingerprinting and based on COI gene sequence data, respectively. Genetic variation within individuals of both populations (WP) played the key role in total genetic variation at species level in surveyed region. Conclusions For the bigfin reef squid species in the surveyed region, the Con Dao population had the higher genetic diversity than the Phu Quoc population, between them existed a low to moderate genetic differentiation and a genetic exchange via gene flow. The DNA fingerprinting data better revealed the genetic differentiation between the two surveyed populations while the mitochondrial COI gene sequence data could show the phylogenetic relationship among the surveyed individuals and the other from the sea regions in Southeast Asia. Based on the results obtained, fisheries management strategies are suggested toward the conservation and sustainable exploitation of this species. [ABSTRACT FROM AUTHOR]