From a cultural point of view, Maize is the most important crop in Mexico, and is also an essential food in the Mexican diet. This study aimed to evaluate the physical and chemical characteristics of maize grain and their influence on the quality of dough and tortillas produced with three native maize cultivars from western Mexico. Physical, structural, and chemical characteristics of the grain were evaluated, including hectolitric weight, 100-grain weight, flotation index, grain structure, grain color, pedicel, pericarp, germ, and floury endosperm, vitreous endosperm, ethereal extract, and protein. Nixtamal and tortilla quality include nixtamal moisture, dough moisture, freshly made tortilla moisture, total soluble solids, pericarp remnant, dough yield, and hot tortilla yield. In general, statistical differences (p ≤ 0.05) were found in almost all variables analyzed. The principal component analysis (PCA) showed that two principal components (PCs), described approximately 79% of the variation, and a positive correlation between the characteristics of freshly made tortilla moisture (FMTM), dough yield (DY), and nixtamal moisture (NM), as well as dough moisture (DM) and hot tortilla yield (HTY). Future studies on these and other native maize cultivars of western Mexico must be conducted to assess the physicochemical and phytochemical characteristics of maize grains that may be useful for food and pharmaceutical applications, contributing to the conservation, propagation, and use of natural and ancient resources.