The South American silver croaker, Plagioscion squamosissimus, represents a species of substantial relevance to artisanal fisheries operating within the sub-middle and lower stretches of the São Francisco River in northeastern Brazil. To elucidate the population structure of this species across its longitudinal gradient, 150 specimens (50 per locality), with standard lengths ranging from 35 to 45 cm, were collected between September 2023 and March 2024 from three fishing associations (Olho D’Água do Casado, Petrolândia, and Rodelas) using gillnets. Body morphometry was examined through two geometric morphometric frameworks based on discrete landmarks: Procrustes coordinates (PCs) and truss network distances (TDs). Otolith morphology was further characterized using two contour-based analytical methods: wavelet transform coefficients (WTCs) and elliptic Fourier descriptors (EFDs). Statistical assessments involved both univariate and multivariate techniques to evaluate morphological variation across sites and sexes. The analyses revealed pronounced secondary sexual dimorphism in otolith morphology, which was absent in overall body shape. Such dimorphism is potentially attributable to sex-specific auditory adaptations related to ecological roles or reproductive behaviors, although this hypothesis warrants further experimental verification. Despite the detected sexual dimorphism, the classification accuracy of individuals to their sampling origin remained remarkably high, ranging from 91% to 94% based on body morphometry (TD and PCs, respectively), and achieving complete (100%) discrimination through otolith contour analyses (EFDs and WTCs). The phenotypic differentiation observed among populations appears to reflect spatially heterogeneous environmental conditions, likely influenced by habitat fragmentation resulting from the construction of two hydroelectric dams that disrupted longitudinal connectivity within the São Francisco River. Overall, the findings indicate that P. squamosissimus populations inhabiting the sub-middle and lower river reaches constitute distinct population units, a conclusion that bears significant implications for the design and implementation of effective management and conservation strategies for the species.