Small mesopelagic fish are ubiquitous in the ocean, representing an important trophic link between zooplankton and tertiary consumers such as larger fish, marine mammals and birds. Lanternfishes (Myctophidae) are common worldwide as well as in the Southern Ocean. However, only 17 of the approximately 250 myctophid species occur exclusively in sub-Antarctic or Antarctic waters. It is unclear whether they colonized these latitudes once and diversified from there, or whether multiple colonization events took place in which multiple ancestral phenotypes entered the Southern Ocean at various times. Phylogeographic patterns have been investigated for individual myctophid species, but so far no study has compared species across the Southern Ocean. Here, we present a dataset with previously unpublished cytochrome c oxidase I (COI; n = 299) and rhodopsin (rh1; n = 87) gene sequences from specimens collected at various locations in the Southern Ocean. Our data extend the DNA barcode library of Antarctic mesopelagic fish substantially. Combined morphological and molecular taxonomy lead to confident species level identification in 271 out of 299 cases, providing a robust reference dataset for specimen identification, independently of incomplete morphological characters. This is highly topical in light of prospective ecological metabarcoding studies. Unambiguous sequences were subsequently combined with publicly available sequences of the global DNA barcode library yielding a dataset of over 1,000 individuals for phylogenetic and phylogeographic inference. Maximum likelihood trees were compared with results of recent studies and with the geographical origin of the samples. As expected for these markers, deep phylogenetic relationships remain partially unclear. However, COI offers unmatched sample and taxon coverage and our results at the subfamily to genus level concur to a large extent with other studies. Southern Ocean myctophids are from at least three distant subfamilies suggesting that colonization has occurred repeatedly. Overall, spatial divergence of myctophids is rare, potentially due to their enormous abundance and the homogenizing force of ocean currents. However, we highlight potential (pseudo-)cryptic or unrecognized species in Gymnoscopelus bolini, Lampanyctus achirus, and the non-myctophid genus Bathylagus.