The Antarctic silverfish Pleuragramma antarcticum (Nototheniidae) is the most abundant pelagic fish inhabiting Antarctic waters. In this study we investigated, through partial sequencing of the D-loop mitochondrial region, samples collected at four different locations in the Southern Ocean, three in the Atlantic and one in the Pacific sector. Sampling was replicated in two different years at two locations. Sequence analysis showed a remarkably high polymorphism, with 110 haplotypes over the 256 investigated specimens, and about 80% of haplotypes occurring only once. Neutrality tests indicated that all samples were not at mutation–drift equilibrium, and suggested a past population expansion. This result was supported by the presence of a star-like topology in the D-loop gene tree, and by results of mismatch distribution. The start of the expansion was dated, using a specifically calibrated clock, between 111 and 126 thousand years ago. This value corresponds to the start of the cooling period that led to the last glaciation peak, and is in close agreement with a recently suggested range expansion for pelagic Antarctic ecosystems. Analysis of molecular variation indicated a small, though highly significant, value of differentiation between samples. This result, together with the lack of association between clades and geographical locations, indicates a weak population structure for the species.