Princeton University Library's rare sixteenth-century polychrome Aztec picture map on deerskin vividly portrays the conversion of the Aztecs to Catholicism during the Early Colonial Period of the Spanish Conquest. A non-invasive technical examination was conducted to identify the colourants and the species of the animal hide support used in the creation of the map. Analysis included spectroscopic techniques such as attenuated total reflectance-Fourier transform infrared spectroscopy and ultraviolet-visible spectroscopy. Light microscopy and UVA fluorescence were also employed as tools. Results of the technical examination found the presence of cochineal, bone black, Maya blue, which are indigenous colourants traditionally associated with Mesoamerica, and a layer of an unknown greenishyellow dye over Maya blue to create the colour green. The examination also discovered what appears to be an orange-yellow gamboge-like substance known as American (sometimes called Mexican) gamboge, which is indigenous to Mexico and parts of Central and South America. Its plant family is Hypericaceae, which is very closely related to Clusiaceae. Clusiaceae is the Southeast Asian and Indian plant family of gamboge, which is commonly used as a pigment in Western, Indian and Asian art. The study's exposure of the obscure substance may spur more research into its use as a colourant of the Mesoamerican palette. The paper also hopes to add to the overall knowledge of Mesoamerican materials in order to better understand aspects of their conservation and preservation.