The genus Macrourus is a small group of bentho-pelagic fishes with three species recognized in the Southern Ocean and one in cold temperate to Arctic waters in the North Atlantic Ocean. DNA barcoding revealed low intra-species (0.0-0.002) and inter-species (0.006-0.020) sequence divergences, with four well supported clades among the three Southern Ocean species. Additional specimens collected in the Ross Sea region, and provisionally identified as M. whitsoni, were divided into two COI haplotypes, based on a Restriction Fragment Length Polymorphism (RFLP) test. Three characters counted/scored in the same Ross Sea region specimens classified individuals into two groups: one with 9 pelvic fin rays, pale body colour, and a single row of relatively long teeth, and a second group with 8 pelvic fin rays, dark body colour, and 2 or more rows of finer teeth, that corresponded with the DNA haplotypes. The COI divergence coupled with the meristic and morphometric differences among specimens provisionally identified as M. whitsoni from the Ross Sea region provided strong evidence for two sympatric species: M. whitsoni pale and M. sp dark. The shallow sequence divergences likely indicate either recent evolutionary divergence or slow nucleotide substitution at COI in this genus.