The exact nature of pigments present in cultured freshwater pearls is still not well known. We examined 21 untreated cultured freshwater pearls from Hyriopsis of typical colors by diffuse reflectance UV-Vis-NIR and Raman scattering measurements, at high resolution. The objective was to establish the relation between color and the nature of the pigment mixture in pearls, using strictly non-destructive methods. All natural color samples show the two major Raman resonance features of unmethylated (unsubstituted) polyenes, not carotenoids. Their general chemical formulae are R-(-CH=CH)N-R′ with N = 6 to 14 and they give absorptions from violet to yellow–green. Each color is due to a mixture of pigments, not a single pigment. Different colors are explained by different mixtures. Each pigment identified by Raman spectroscopy can be related to a specific absorption with apparent maximum in the range 405–568 nm, thus absorbing from violet to yellow–green. This is the first study of the precise relation between Raman and diffuse reflectance spectra of cultured freshwater pearls.