Introduction The tomato plant, Solanum lycopersicum L. (Solanaceae), is one of the most widely consumed vegetables in the world and plays an important role in human diet. Tomato cultivars are hosts for diverse types of pests, implying diverse chemical defence strategies. Glycoalkaloids are the main specialised metabolites produced by tomato leaves and fruits to protect against pests. However, the roots have received little attention, leading to limited knowledge about their phytochemical content. Objective The main goal of the current study was the development of an untargeted ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) based metabolomic approach to study phytochemical variations in tomato roots at two different development stages (i.e. 34th and 62nd day after sowing). Methods UHPLC-HRMS was used to establish the fingerprint of 24 batches of tomato roots. Statistical analyses were performed to highlight the compounds that discriminated between young and mature tomato roots. A dereplication strategy using molecular networking and HRMS/MS data was set up to identify the metabolites regulated during early root development. Key findings The main biomarkers were guanidine and adenosine derivatives associated with tryptophan. Secondary metabolites such as glycoalkaloids and steroidal alkaloids were also characterised. Most of the metabolites were up-regulated in young tomato roots (34 days old) while tryptophan was up-regulated in the older roots (62 days old). Conclusion The metabolic changes observed in this work contribute to a deeper understanding of early-stage root development and may help our understanding of the complex processes involved in the tomato root defence arsenal.