Abstract: To investigate the differences in metabolic profile characteristics of Thlaspi arvense seeds between Shandong and Gansu, this study employed an integrated analysis approach combining metabolomics and lipidomics. Two superior materials from Gansu (GS) and Shandong (SD), screened from five Thlaspi arvense resources collected from different regions in China, were subjected to identification and comparative analysis of their seed metabolic profile characteristics. Untargeted analysis has detected 645 metabolites, dominated by lipids/lipid-like molecules, phenylpropanoids/polyketides, and organic acid derivatives. Targeted lipidomics has identified 1,133 lipid species, primarily glycerolipids, glycerophospholipids, and glycolipids, with triacylglycerols (TG) as the core component. Through principal component analysis (PCA), orthogonal partial least squares-discriminant analysis (OPLS-DA), and metabolic pathway analysis, 21 differentially accumulated metabolites and 112 differentially accumulated lipids have been identified (OPLS-DA VIP > 1, P value < 0.05, FC > 1 or < 1). GS pennycress showed significantly upregulated lipid accumulation, enriching medium- to long-chain unsaturated fatty acids and demonstrating broader improvement potential in TG biosynthesis. By contrast, SD pennycress prioritized the accumulation of medicinal secondary metabolites (e.g., tannins, alkaloids), consistent with KEGG enrichment results. This study therefore provides critical insights for the functional food development and genetic improvement of pennycress seeds.