The marine silverside (Odontesthes argentinensis) is an euryhaline species, distributed along the southwest coast of the Atlantic Ocean, present in estuaries, brackish coastal lagoons and shallow marine waters. It is a significant economic resource for local fisheries in southern Brazil, Uruguay and Argentina. The aim of this work was to contribute to knowledge on the stock spatial structure of the silverside, using otolith shape analysis, based on samples from nine locations in the Argentinian Sea, covering a large distribution range of the species. A combination of elliptic Fourier descriptors, Wavelet coefficients and otolith Shape indices were explored by multivariate statistical methods. The application of wavelet and combined wavelet, Fourier and Shape Indices were the most effective variables to discriminate between sampling sites (7.42 total error). PERMANOVA analysis of otolith shape revealed multivariate significant differences between north versus south locations (p < 0.0001). The results obtained show that the spatial structure of O. argentinensis presents a North–South gradient with marked differences between the extreme localities of the north (Mar del Plata, Quequén) with more elliptical shapes than those in the south (San Blas, San Antonio Este) and an isolated group conformed by Puerto Lobos.