A large number of metabolites are found in each plant, most of which have not yet been identified. Development of a methodology is required to deal systematically with unknown metabolites, and to elucidate their biological roles in an integrated ‘omics’ framework. Here we report the development of a ‘metabolite annotation’ procedure. The metabolite annotation is a process by which structures and functions are inferred for metabolites. Tomato (Solanum lycopersicum cv. Micro-Tom) was used as a model for this study using LC-FTICR-MS. Collected mass spectral features, together with predicted molecular formulae and putative structures, were provided as metabolite annotations for 869 metabolites. Comparison with public databases suggests that 494 metabolites are novel. A grading system was introduced to describe the evidence supporting the annotations. Based on the comprehensive characterization of tomato fruit metabolites, we identified chemical building blocks that are frequently found in tomato fruit tissues, and predicted novel metabolic pathways for flavonoids and glycoalkaloids. These results demonstrate that metabolite annotation facilitates the systematic analysis of unknown metabolites and biological interpretation of their relationships, which provide a basis for integrating metabolite information into the system-level study of plant biology.