In an effort to understand the mechanism of otolith elemental incorporation, the distribution of strontium (Sr) and sulfur (S) in otoliths of Platycephalus bassensis was investigated in conjunction with otolith growth patterns. Optimisation of electron probe microanalysis (EPMA) quantitative mapping achieved both high spatial resolution (<3μm) and two-dimensional visualisation of the fine scale Sr and S distributions in otoliths of P. bassensis with minimal damage. Electron backscatter diffraction (EBSD) mapping confirmed that grain growth is aligned with the otolith c-axis, with grain orientation independent of both otolith elemental composition and growth patterns. Results showed a linear correlation between Sr and S distribution (R2≤0.86), and a clear association with the otolith growth patterns determined by scanning electron microscopy. Further examination by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) showed that incorporation of Mg and Ba appeared independent of both S distribution and the growth patterns. The results suggest that element incorporation into the otolith is linked to the organic composition in the endolymph during mineralisation, and the organic matrices may assist, in part, the uptake of Sr. Thus, these findings may have significant implications for the interpretation of otolith Sr chemistry.