Although dark septate fungal endophytes (DSE) occur widely in association with plant roots in cold-stressed habitats, little is known of the taxonomic status of DSE in Antarctica. Here we investigate the phylogenetic affinities of DSE colonising the roots of Colobanthus quitensis and Deschampsia antarctica, the two maritime Antarctic vascular plant species. Two hundred and forty-three DSE were isolated from roots collected from 17 sites across a 1470 km transect through maritime and sub-Antarctica. The ITS1-5.8S-ITS2 nuclear ribosomal gene cluster of representative isolates was sequenced, and the sequences were recovered in 10 sequence groups and sub-groups. Nine of the sequence groupings could be placed in the Helotiales and the remaining one showed high homology to a large number of currently unassigned anamorphic ascomycete sequences. Of the Helotiales, Leptodontidium orchidicola, Rhizoscyphus ericae and species of Tapesia and Mollisia could be confidently identified. This study demonstrates that members of the Helotiales, including several widely-recognised DSE genera, commonly colonise the roots of C. quitensis and D. antarctica in the Antarctic.