The number of studies using otolith chemistry as a tool to reconstruct the environmental history of fishes and to detect population structure continues to rise, despite the fact that factors influencing otolith deposition are not fully understood. Many studies have examined the influence of environmental parameters on otolith composition, but none to date have tested the possible influence of intrinsic factors. Using lab broodstock populations, we examined the influence of genetics and temperature on Mg:Ca, Mn:Ca, Sr:Ca, and Ba:Ca concentrations and partition coefficients in the otoliths of juvenile Atlantic silversides, Menidia menidia . Fish from two populations, South Carolina, USA, and Nova Scotia, Canada, were reared in 15, 21, and 27 °C. We found significant (p < 0.05) differences in otolith Mg:Ca, Mn:Ca, and Ba:Ca ratios as well as in Mg, Mn, and Ba partition coefficients among populations. Such genetic influences on otolith elemental concentrations have important implications for understanding the physiological mechanisms underlying otolith deposition and enhance the utility of otolith chemistry as a marker of fish population structure in the wild.