ABSTRACT: Estuaries provide critical habitat, food, and refuge for juvenile fish. Elemental concentrations in otoliths can retrospectively identify estuarine nursery habitats that contribute disproportionately to adult populations, providing valuable information for fisheries and coastal ecosystems. The present study aimed to (1) compare elemental signatures of juvenile (age 0+) sand flounder Rhombosolea plebeia otoliths collected from 9 estuaries in east Otago, New Zealand, and (2) assess the application of otolith microchemistry for identifying the nursery habitats contributing to adult (age 1+) sand flounder from the coastal shelf population. Otoliths from juvenile flounder were collected in November 2020, and adults of the same cohort were collected in the winter/spring of 2022. Laser ablation inductively coupled plasma mass spectrometry quantified 12 element concentrations within the post-settlement region of juvenile and adult otoliths using depth-profiling and ablation transects, respectively. Significant differences in post-settlement elemental concentrations were detected among estuaries. Juvenile and adult sand flounder were classified to their nursery origin using canonical analysis of principal coordinates. Grouping estuaries by the New Zealand Estuary Trophic Index classification scheme improved the overall juvenile classification accuracy from 56.7% (51/90) to 82.2% (74/90). Adult sand flounder (n = 78) were classified to have originated from shallow intertidal dominated estuaries (62%), followed by Otago Harbour (32%) and shallow, short residence time river and tidal river with adjoining lagoon estuaries (6%). The results highlight how otolith microchemistry can provide information on the use of critical estuarine nursery habitats for a commercially important species.