In the present study, the population differentiation of the purpleback flying squid () in the South China Sea was examined based on mitochondrial ND2, COI, and 16S rDNA sequences, using samples collected between September 2012 and September 2015. Phylogenetic analysis revealed two significantly divergent monophyletic groups corresponding to the "medium form" and the "dwarf form, " but no geographical lineages corresponding to the sampling localities or seasonal groups were found. The Analysis of Molecular Variance also revealed significant genetic differentiation between the "medium form" and the "dwarf form" (<0.0001), while no genetic variance between either geographic populations or seasonal groups was found. The genetic diversity analysis showed that both the "medium form" and the "dwarf form" of in the South China Sea were characterized by high haplotype diversity (0.9465 and 0.8545) and low nucleotide diversity (0.0051 and 0.0021). The genetic distance between the ‘medium form’ and the "dwarf form" based on ND2, COI, and 16S sequences were 14.0%, 9.6%, and 8.8%, respectively. The genetic distances between the two forms were considerably larger than the genetic distances within each of the two forms (<1%). It was demonstrated that two divergent populations of were sympatrically distributed in the South China Sea, i.e. the "medium form" and the "dwarf form." The genetic differentiation between these two populations was similar to that among separate species within Ommastrephidae, which indicated that the two morphologically distinct forms of the purpleback flying squid, namely the "medium form" (middle sized, with a photophore) and the "dwarf form" (small sized, without a photophore), are two distinct species.