Cassava is a significant food security crop in several developing countries. Metabolites in cassava roots provide numerous nutrients essential for human health. Exploiting the diversity of nutritional ingredients present in cassavas is vital for improving its nutritional value. To address this problem, root metabolomes of three cassava cultivars with white-flesh, light-yellow-flesh and yellow-flesh were comprehensively measured, respectively. A total of 508 metabolites were detected in cassava roots, including 300 primary metabolites and 185 secondary metabolites. There were 22.6% to 34.1% metabolites exhibiting significant variations among the three cassava cultivars. The light-yellow-flesh cassava contained higher contents of secondary metabolites, especially flavone, phenylpropanoids and alkaloids, and lower contents of primary metabolites except lipids, alcohols, vitamins and derivatives. Compared with light-yellow-flesh cassava, the yellow-flesh cassava contained higher contents of amino acid and derivatives, but lower contents of phenylpropanoids, nucleotide and derivates. White-flesh cassava contained higher contents of primary metabolites, especially amino acid and derivatives, but lower contents of secondary metabolites except flavonoid and indole derivatives. Transcriptome analyses were parallelly performed to decipher the potential mechanisms regulating the accumulations of related metabolites. Several pathways were both enriched by differentially expressed genes and differentially accumulated metabolites, supporting that metabolisms of these metabolites were regulated at transcriptional level. These results expand the knowledge on metabolite compositions in cassava roots and provide substantial information for genetic improvement of cassavas with high nutritional values. © 2021 The Authors