We investigated the relationships between the opacity and the physico-chemical characteristics of fish otoliths and more specifically their aragonite and organic fractions. The analysis of these two fractions on otolith macrostructures was performed using Raman microspectrometry on both translucent and opaque zones of otoliths of pollock (Pollachius virens) and European hake (Merluccius merluccius). The magnitude of the Raman signatures of the aragonite and organic fractions were strongly correlated to otolith opacity with maxima in translucent zones. Opacity models, built from Raman signatures, successfully predicted the observed opacity for both species. A partial decorrelation of different aragonite signatures between translucent and opaque zones was revealed and discussed in terms of organisation (size, orientation) of aragonite crystals. Two categories of organic signatures with opposite effects on the opacity were identified, suggesting differences in organic compounds and (or) variations in their relative quantities. These original contributions provided new insight for understanding otolith biomineralization mechanisms as well as for interpreting and discriminating otolith macrostructures.