In order to explore the cause for the existence of variegated substance in seawater cultured pearls,the authors observed the microstructure of variegated substance with scanning electron microscopy (SEM), and then used thermogravimetric analyzer and Fourier infrared spectrometer (FIR) to analyze the composition of variegated substance. Through SEM observation, the authors found that the variegated seawater cultured pearl contains nacre, prismatic layer, intermediate layer, variegated layer and nucleus from the outer part to the inner part. The thickness of the prismatic layer and the intermediate layer are 20 and 10 μm, respectively, whereas the thickness of variegated layer is nonuniform, even reaching 50 μm. The experimental result shows that the variegated substance mainly consists of aragonite and proteins which jointly constitute the laminated and reticular structure.