Introduction: Lettuce (Lactuca sativa L.) is generally not specifically acknowledged for its taste and nutritional value, while its cultivation suffers from limited resistance against several pests and diseases. Such key traits are known to be largely dependent on the ability of varieties to produce specific phytochemicals. Objectives: We aimed to identify promising genetic resources for the improvement of phytochemical composition of lettuce varieties. Methods: Phytochemical variation was investigated using 150 Lactuca genebank accessions, comprising a core set of the lettuce gene pool, and resulting data were related to available phenotypic information. Results: A hierarchical cluster analysis of the variation in relative abundance of 2026 phytochemicals, revealed by untargeted metabolic profiling, strongly resembled the known lettuce gene pool structure, indicating that the observed variation was to a large extent genetically determined. Many phytochemicals appeared species-specific, of which several are generally related to traits that are associated with plant health or nutritional value. For a large number of phytochemicals the relative abundance was either positively or negatively correlated with available phenotypic data on resistances against pests and diseases, indicating their potential role in plant resistance. Particularly the more primitive lettuces and the closely related wild relatives showed high levels of (poly)phenols and vitamin C, thus representing potential genetic resources for improving nutritional traits in modern crop types. Conclusion: Our large-scale analysis of phytochemical variation is unprecedented in lettuce and demonstrated the ample availability of suitable genetic resources for the development of improved lettuce varieties with higher nutritional quality and more sustainable production.