Yellow shell morphotypes of the pearl oyster Pinctada fucata martensii are rare and intriguing, whereas polyene pigments in mineralized shells from yellow pearl oysters have not been identified. An in situ micro-Raman spectroscopy and scanning electron microscopy investigation were performed on yellow and brown shell pearl oysters. Carotenoids or polyene pigments were found in both prismatic and nacreous layer from yellow pearl oysters. The peaks of pigments in the yellow prismatic surface were at wavenumbers of 1,280.8, 1,318.7, 1,146.1, and 1,577.2 cm^–1, whereas those in the nacreous layer were at 1,135.3 and 1,530.6 cm^–1. In brown pearl oysters, carotenoids related vibration peaks were not found in the prismatic layer, whereas slight spectra in the nacreous layer at wavenumbers of 1,136.3 and 1,531.4 cm^–1 were attributed to polyene pigments. Pigments in the prismatic and nacreous layers of the yellow shell were not the same, and the former showed shorter polyene backbones and more complex molecular structures. This research provided characteristic Raman spectra for brown and yellow shell phenotypes of pearl oysters and can contribute to explore the connection between polyene pigments and biomineralization.