Abstract: In the pursuit of a comprehensive understanding of the metabolic distinctions between red wolfberry (Lycium barbarum L.) and yellow wolfberry (Lycium barbarum L. var. auranticarpum K.F.Ching), an analytical approach integrating ultra-performance liquid chromatography with tandem high-resolution mass spectrometry was employed, alongside a broadly targeted metabolomics strategy. This methodology facilitated the identification of a total of 202 differential metabolites in the positive ion mode, with 148 displaying up-regulated expression and 54 showing down-regulated expression. Similarly, in the negative ion mode, 240 differential metabolites were detected, with 183 up-regulated and 57 down-regulated, under stringent criteria of ≥ 2-fold or ≤ 0.5-fold change, a variable importance in the projection (VIP) score of ≥ 1, and a significance level of P<0.05. Subsequent KEGG pathway enrichment analysis revealed that these differential metabolites were predominantly associated with secondary metabolite biosynthesis, including tryptophan, glucosinolate, valine, isoleucine, leucine, and flavonoid pathways. The data suggested that the differential metabolites in Lycium barbarum L. and Lycium barbarum L. var. auranticarpum K.F.Ching were predominantly flavonoids, alkaloids, phenolic acids, amino acids, and their derivatives, with a general trend of up-regulation. These metabolites may be linked to the growth, development, disease resistance, and nutritional value of Lycium barbarum L. var. auranticarpum K.F.Ching, potentially through their involvement in tryptophan metabolism and biosynthesis pathways for valine, leucine, isoleucine, and flavonoids. The objective of this study was to contribute to the existing knowledge base on the nutritional value of these two wolfberry species and to offer a theoretical foundation for the refinement of wolfberry-related products through extensive metabolomics profiling.