ABSTRACT: Reconstructing the movements of fish among different environments and incorporating patterns of spatial population structure contribute to improved accuracy in the assessment of marine fishery resources. The southern flounder Paralichthys lethostigma is a valuable flatfish throughout its range in the US South Atlantic and Gulf of Mexico, but stock conservation and management is hindered by critical knowledge gaps related to patterns of movement and the level of mixing among populations. Identifying stock structure and connectivity can be challenging in species with complex life histories, and otolith geochemical signatures have been effectively used as natural markers to estimate population connectivity in migratory fishes. With this approach, we inferred the degree of exchange of southern flounder among broad US South Atlantic regions by predicting the nursery origins of adults captured in North Carolina and South Carolina estuaries. Baseline nursery profiles were first established by analyzing stable isotopes (δ¹³C, δ¹⁸O) and trace elements (Mg:Ca, Mn:Ca, Sr:Ca, Ba:Ca) sampled from juvenile southern flounder otoliths. Quadratic discriminant analysis discriminated among state-scale nursery regions (North Carolina, South Carolina, and Florida) with 72% cross-validation accuracy. Adult southern flounder from the same cohort were subsequently classified to the atlas of nursery signatures obtained from the juvenile fish using a maximum likelihood mixed stock analysis. Results revealed a lack of nursery-state fidelity and the potential for broad-scale movement of post-migratory adults along the US South Atlantic coast, which will contribute to the definition of appropriate spatial scales for management.