The use of natural tags for identifying the source locations of individuals recruited to fish populations has become an important area of research in fisheries science. We used stable carbon (δ¹³C) and oxygen (δ¹⁸O) isotope ratios in otoliths to differentiate juvenile winter flounder (Pseudopleuronectes americanus) collected from locations along the coast of Rhode Island, USA. Sagittal otoliths from the flounder were analysed for δ¹³C and δ¹⁸O using isotope ratio mass spectrometry. Plots of δ¹³C v. δ¹⁸O and discriminant function analysis (DFA) indicated differences among samples from Narragansett Bay, the coastal ponds and the Narrow River. Separations were observed among fish from the upper, middle and lower portions of Narragansett Bay and along the salinity gradient in Narrow River. Otoliths from sites grouped on the basis of low, medium and high salinities also showed differences according to DFA. Correlation analysis indicated a significant negative relationship (r = –0.60, P = 0.009) between seawater temperature and δ¹⁸O, and a significant positive relationship (r = 0.93, P < 0.001) between salinity and δ¹⁸O for the combined dataset. These results indicate that δ¹³C and δ¹⁸O values of juvenile winter flounder otoliths may prove useful for differentiating individuals from various juvenile habitats.