The Trematominae, a subfamily of the Nototheniidae, are typical of the high-Antarctic shelf waters. Within the Trematominae examples of phenotypic plasticity and possible cryptic speciation have been observed. Morphological identification of adult stages can be problematic in cases of high phenotypic plasticity or cryptic speciation. Additionally, postlarval and juvenile stages often have traits still under development and which lack distinction. A microsatellite DNA multiplex of six markers has been developed for Trematomus newnesi (Van Houdt et al. 2006). This multiplex was tested on five additional trematomid taxa: Pagothenia borchgrevinki, Trematomus bernacchii, Trematomus eulepidotus, Trematomus hansoni and Trematomus scotti. We used these six microsatellite loci to assess the genetic differentiation among species and the resolution power of these loci for individual-based assignment methods. The six species could be well discriminated by conventional methods such as principal component analysis and distance-based methods, and individual Bayesian assignment methods. This marker set can be used for a number of purposes, including the identification of eggs and larval and adult stages. It is also useful for the investigation of recent phylogenetic patterns, as well as the detection of cryptic speciation, which has been suggested for T. bernacchii and T. newnesi but never confirmed with high polymorphic genetic markers.