Retrospective determination of the early life history of fish using the microelemental analysis of their otoliths is dependent upon understanding the factors that affect this elemental composition. Here, juvenile Atlantic croaker (Micropogonias undulatus) were reared under different treatments of temperature and salinity to determine their impacts on elemental inclusion rates in otoliths. Solution-based inductively coupled plasma mass spectrometry (ICPMS) was used to measure 21 isotopes in each otolith: isotopic concentrations ranged over seven orders of magnitude, and differed significantly amongst the temperature–salinity regimes. Univariate analyses identified 13 isotopes that contributed to these multivariate differences; the influence of temperature was stronger than that of salinity. Within each treatment there was a significant relationship between otolith microchemistry and otolith size. To some extent this confounded the interpretation of the between-treatment effect of temperature. In contrast, both the otolith and somatic growth rates were similar between the two salinity treatments, indicating that differences in elemental fingerprints were unambiguously related to the salinity difference, probably a response to the elemental concentrations in the tank water. Overall the study highlighted the current poor understanding of the mechanism of contamination of otoliths by trace elements and their incorporation into the otolith microstructure.