Abstract: Trapa, a genus of aquatic macrophytes traditionally used in Chinese medicine and food, holds substantial ecological and economic significance. The pericarps of Trapa species are particularly enriched in polyphenols, contributing to notable anticancer and antioxidant activities. This study investigated the pharmacophylogenetic relationships among seven representative Trapa species by integrating secondary metabolite profiling with comparative chloroplast genomics. The selected species included one cultivated species (T. acornis Nakano) and six wild species (T. maximowiczii Korsh., T. quadrispinosa Roxb., T. bispinosa Roxb., T. manshurica Flerow, T. kozhevnikovirum Pshennikova, and T. japonica Flerow). Broad-targeted metabolomic profiling of pericarp extracts was conducted using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Concurrently, phylogenetic relationships were inferred using maximum-likelihood (ML) and Bayesian inference (BI) analyses based on the complete chloroplast genomes. A total of 273 secondary metabolites were identified across the seven Trapa species, with marked interspecific variation and strong intraspecific consistency in metabolic profiles. Notably, clustering patterns based on metabolite composition closely mirrored those derived from chloroplast phylogenomics, indicating that species with closer genetic relationships exhibited greater similarity in secondary metabolite profiles. These findings provide a valuable scientific foundation for the effective exploration and sustainable utilization of Trapa medicinal resources.