Abstract Accurate species identification is essential to assess biodiversity and species richness in ecosystems threatened by rapid and recent environmental changes, such as warming in most Antarctic waters. The Lepidonotothen species complex comprises demersal notothenioid fishes which inhabit the shelf areas of the Antarctic Peninsula, the Scotia Arc and sub‐Antarctic islands with a circum‐Antarctic distribution. Species determination in this group has often been problematic. In particular, whether Lepidonotothen squamifrons and Lepidonotothen kempi are valid as separate species has been questioned. In this study, we analysed the genetic variation among four nominal southern polar species within this complex ( L. kempi , L. squamifrons , Nototheniops larseni , Nototheniops nudifrons ) by means of three different markers ( ND2 and tRNA mitochondrial genes and a panel of 16 nuclear microsatellites). We tested whether individuals morphologically assigned to L. kempi showed genetic separation from L. squamifrons . Our analyses indicated a lack of differentiation between L. kempi and L. squamifrons . However, a genetically distinct population was found for L. squamifrons at the Shag Rocks islands near South Georgia. Antarctic and sub‐Antarctic islands are known to be home to many cryptic species and further studies will elucidate if the genetically differentiated population we found potentially originated from this context and can be considered an incipient species. Our analysis contributes to further characterize the species composition of the most abundant fish suborder in the Southern Ocean, which is among the regions most threatened by climate change.