The biocrystallization process and the prescence of manganese in sagitta otoliths is described for the first time via electron paramagnetic spectroscopy. Spectra of Mn²⁺ are applied to verify inter-species differences in 12 Sciaenidae species and geographic variations among three stocks of banded croaker Paralonchurus brasiliensis and rake stardrum Stellifer rastrifer. Similar spectra shape and zero-field splitting D (22.4 mT) indicated that the biocrystallization process is the same for all fish species, at any stage of development and under different environmental conditions, corresponding to Mn²⁺ in the aragonite (CaCO₃). Inter-species differences in Mn²⁺ concentration were detected regarding the otolith shape. Elongated otoliths have higher manganese concentrations compared to rounded otoliths. Geographic variations among stocks of P. brasiliensis and S. rastrifer could be detected. Fish stocks under riverine influence have less Mn²⁺ concentration than ones under marine influence. Manganese detection via electron paramagnetic spectroscopy is a non-destructive approach that can be useful in fish stocks studies.