The Afrasiab murals discovered in the northeast of Samarkand, Uzbekistan—the center of the ancient Silk Road—are presumed to date to the mid-seventh century during the Sogdian era. Although previous studies have examined the primary materials of the pigments used in these murals using chemical and microscopic analyses, in-depth investigations of the pigment raw material composition have not been conducted to verify the results of these studies. We applied X-ray diffractometry, Raman spectroscopy, and scanning electron microscopy in conjunction with energy-dispersive X-ray spectroscopy for the first time to identify the raw materials of ancient pigments in fragments obtained from the Afrasiab murals. The results show that lazurite, cinnabar, and amorphous carbon were used as blue, red, and black pigments, respectively. Moreover, we identified that pigments were not directly painted on the wall surface; instead, they were painted on a white undercoat of gypsum plaster, similar to other ancient Silk Road wall paintings. The results of this study can benefit the provision of more accurate information with regard to the composition of raw materials and further support the selection of appropriate substances for the purposes of conservation and restoration of Afrasiab murals.