Manihot esculenta roots rich in β-carotene are an important staple food for populations with risk of vitamin A deficiency. Cassava genotypes with high provitamin A activity have been identified as a strategy to reduce the prevalence of deficiency of this vitamin, In this study, the metabolomics characterization focusing on the carotenoid composition of ten cassava genotypes cultivated in southern Brazil by UV-visible scanning spectrophotometry and reverse phase-high performance liquid chromatography was performed. The data set was used for the construction of a descriptive model by chemometric analysis. The genotypes of yellow roots were clustered by the higher concentrations of cis-β-carotene and lutein. Inversely, cream roots genotypes were grouped precisely due to their lower concentrations of these pigments, as samples rich in lycopene differed among the studied genotypes. The analytical approach (UV-Vis, HPLC, and chemometrics) used showed to be efficient for understanding the chemodiversity of cassava genotypes, allowing to classify them according to important features for human health and nutrition.