Roots, tubers and bananas (RTB) are vital staples for food security in the worlds' poorest nations. A major constraint to current RTB breeding programmes is limited knowledge about the available diversity due to lack of efficient germplasm characterisation and structure. In recent years large-scale efforts have begun to elucidate the genetic and phenotypic diversity of germplasm collections and populations and yet, biochemical measurements have often been overlooked despite metabolite composition being directly associated to agronomic and consumer traits. Herein we present a compound database and concentration range for metabolites detected in the major RTB crops: banana (Musa spp.), cassava (Manihot esculenta), potato (Solanum tuberosum), sweetpotato (Ipomoea batatas), and yam (Dioscorea spp.), following metabolomics-based diversity screening of global collections held within the CGIAR institutes. The dataset including 711 chemical features provides a valuable resource regarding the comparative biochemical composition of each RTB crop and highlights the potential diversity available for incorporation into crop improvement programmes. Particularly, the tropical crops cassava, sweetpotato and banana displayed more complex compositional metabolite profiles with representations of up to 22 chemical classes (unknowns excluded) than that of potato, for which only metabolites from 10 chemical classes were detected. Additionally, over 20% of biochemical signatures remained unidentified for every crop analysed. Integration of metabolomics with the on-going genomic and phenotypic studies will enhance omics-wide associations of molecular signatures with agronomic and consumer traits via easily quantifiable biochemical markers to aid gene discovery and functional characterisation.