Squid of the genus Lolliguncula Steenstrup, 1881 are small bodied, coastal species capable of tolerating low salinity. Lolliguncula sp. are found exclusively in the New World, although only one of the four recognized species (Lolliguncula brevis) occurs in the Atlantic Ocean. Preliminary morphological analyses suggest that Lolliguncula brevis populations in the North and South Atlantic may represent distinct species. The principal objective of the present study was to verify the phylogenetic relationships within the genus and test for the presence of possible cryptic species. Both gene and species tree topologies indicated that Lolliguncula brevis specimens from the North and South Atlantic represent distinct phylogenetic clades. In contrast with previous studies, L. panamensis was identified as the basal species of the genus. Our results provide important insights into the phylogenetic relationships among the Lolliguncula specimens analyzed, and confirm the genetic separation of Lolliguncula brevis populations of the North and South Atlantic at the level of sister species.