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A prismatic layer is present in both freshwater-cultured pearls and seawater-cultured pearls. The phase structure of the prismatic layer of freshwater-cultured pearls is investigated for the first time and novel findings are reported in this paper. X-ray diffraction (XRD), optical microscopy (OM) and micro-infrared (IR) spectroscopy were used to characterize the crystallized layers of high and low-quality freshwater-cultured pearls. Results show that there is no calcite in the prismatic layer of freshwater-cultured pearls, which is different from what was found for seawater-cultured pearls. We found that an aragonite phase was present in the prismatic layer of high-quality freshwater-cultured pearls and that a vaterite or aragonite phase was present in low-quality pearls. Based on this study an internal factor that influences the quality of freshwater-cultured pearls is discussed.