The present study applied Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS) on a large number of natural and cultured pearls from saltwater and freshwater environments, which revealed that freshwater (natural and cultured) pearls contain relatively higher quantities of manganese (Mn) and barium (Ba) and lower sodium (Na), magnesium (Mg) and strontium (Sr) than saltwater (natural and cultured) pearls. A few correlations between the host animal’s species and chemical elements were found; some samples from Pinctada maxima (P. maxima) are the only studied saltwater samples with 55Mn >20 ppmw, while some P. radiata are the only studied saltwater samples with 24Mg <65 ppmw and some of the P. imbricata are the only studied saltwater samples with 137Ba >4.5 ppmw. X-ray luminescence reactions of the studied samples has confirmed a correlation between its yellow-green intensity and manganese content in aragonite, where the higher Mn2+ content, the more intense the yellow-green luminescence becomes. Luminescence intensity in some cases is lower even if manganese increases, either because of pigments or because of manganese self-quenching. X-ray luminescence can be applied in most cases to separate saltwater from freshwater samples; only samples with low manganese content (55Mn <50 ppmw) might be challenging to identify. One of the studied natural freshwater pearls contained vaterite sections which react by turning orange under X-ray due to a different coordination of Mn2+ in vaterite than that in aragonite.