show Abstracthide AbstractNotothenioids are a clade of ~120 species of marine fishes distributed in extreme southern hemisphere temperate near-shore habitats and in the Southern Ocean surrounding Antarctica. Over the past 25 years, molecular and morphological approaches have redefined hypotheses of relationships among notothenioid lineages as well as their relationships among major lineages of percomorph teleosts. These phylogenies provide a basis for investigation of mechanisms of evolutionary diversification within the clade, and have enhanced our understanding of the notothenioid adaptive radiation. Despite extensive efforts, there remain several questions concerning the phylogeny of notothenioids. In this study we deploy DNA sequences of ~100,000 loci obtained using RADseq to investigate the phylogenetic relationships of notothenioids and to assess the utility of RADseq loci for lineages that exhibit divergence times ranging from the Paleogene to the Quaternary. The notothenioid phylogenies inferred from the RADseq loci provide unparalleled resolution and node support for several long standing problems including, 1) relationships among species of Trematomus, 2) resolution of Indonotothenia cyanobrancha as the sister lineage of Trematomus, 3) the deep paraphyly of Nototheniidae, 4) the paraphyly of Lepidonotothen s.l., 5) paraphyly of Artedidraco, and 6) the monophyly of the Bathydraconidae. Assessment of site rates demonstrates that RADseq loci are similar to mtDNA protein coding genes and exhibit peak phylogenetic informativeness at the time interval during which the major Antarctic notothenioid lineages originated and diversified. In addition to providing a well-resolved phylogenetic hypothesis for notothenioids, our analyses quantify the predicted utility of RADseq loci for Cenozoic phylogenetic inferences.