The use of preserved otoliths for stable isotope analysis assumes handling and preservation procedures do not alter the isotopic composition of the otolith. Otoliths from wild and hatchery-reared salmonids (brook charr, Salvelinus fontinalis, and Atlantic salmon, Salmo salar) were used to test for possible δ18O preservation effects in ethanol and formalin preservation experiments at varying temperatures. Analysis of variance (ANOVA) demonstrated a significant interaction effect between species and preservative during preservation. Possible causes for the observed effect are discussed in relation to species-specific differences in otolith chemistry related to growth and environment including: (1) chemical mechanisms of dissolution-recrystallisation involving the precipitation of secondary minerals within and at the otolith surface; (2) adsorption of ions at available binding sites on the otolith surface; and (3) isotopic exchange during otolith surface dissolution and/or reprecipitation processes. Differential occurrence of vaterite and aragonite in otoliths is believed to account for some of the observed effects as a result of otolith density differences. Isotopic exchange is also argued to cause much of the observed variation in species-specific preservation effects. Biologically, study findings imply that preserved otoliths should not be used for baseline paleoclimatic or individual fish thermal reconstructions, or the development of δ18O-fractionation equations, without the prior use of pilot studies to determine preservation effects. Copyright © 2007 John Wiley & Sons, Ltd.