The objectives of this study were to characterize relationships between water and paddlefish Polyodon spathula dentary Sr:Ca, δ18O and stable hydrogen isotope ratio (δD) to determine the accuracy with which individual P. spathula could be assigned to their collection locations using dentary-edge Sr:Ca, δD and δ18O. A laboratory experiment was also conducted to determine whether dentary Sr:Ca in ... [Show full abstract] age 0 year P. spathula would reflect shifts in water Sr:Ca to which fish were exposed. Significant linear relationships between water and dentary Sr:Ca, δD and δ18O were observed, although the relationship between water and dentary δ18O was weaker than those for Sr:Ca and δD. Classification success for individual fish to collection locations that differed in water Sr:Ca, δD and δ18O ranged from 86 to 100% based on dentary-edge Sr:Ca, δD and δ18O. Dentary Sr:Ca increased significantly in laboratory-reared age 0 year P. spathula following 4 weeks of exposure to elevated water Sr:Ca; dentary Sr:Ca of fish held in water with elevated Sr:Ca was also significantly higher than that of control fish reared in ambient laboratory water. Results indicated that P. spathula dentaries reflect water signatures for commonly-applied natural chemical markers and strongly suggest that dentary microchemistry and stable-isotopic compositions will be applicable for reconstructing P. spathula environmental history in locations where sufficient spatial differences in water chemistry occur.