The thread herring Opisthonema oglinum (Lesueur, 1818) is a small pelagic fish distributed in the western margin of the Atlantic Ocean from the United States to Argentina. It is a target species for diverse commercial fisheries, including the Brazilian industrial purse seine fleets that operate in the Southeastern Brazilian Bight. To investigate the geographic variation in the thread herring populations in this fishing ground, sagittal otoliths were collected from two areas: Rio de Janeiro (RJ: 23°04′ S, 44°03′ W) and Santa Catarina (SC: 26°05′ S, 48°18′ W). Otolith shape analyses and multi-elemental signatures were statistically evaluated using elliptical Fourier descriptors and elemental/Ca ratios. Remarkable differences in the thread herring otoliths between the two areas were found. The previous scenario in which the thread herring constitutes a single panmictic population in the Atlantic Ocean is now debatable. The implications of these results in terms of rational fisheries management for this species are highlighted.