Abstract: The diphenyl ether herbicide fomesafen is the main herbicide used to control Amaranthus retroflexus L. in soybean fields. However, the resistance of A. retroflexus to fomesafen in Heilongjiang Province is increasingly, significantly impacting soybean yields. Currently, there is no metabolomics research available on the resistance mechanism of A. retroflexus to fomesafen. In this study, the metabolomics analysis of leaf tissues from the fomesafen sensitive populations (SY) group, resistant populations (RY) group, and untreated (CK) group was performed. Principal Component Analysis (PCA) and Orthogonal Partial Least-squares Discrimination Analysis (OPLS-DA) results showed that the metabolite profile of the sample from RY group was closer to that from CK group. Kyoto Gene and Genome Encyclopedia (KEGG) enrichment analysis and topology analysis revealed that three main pathways are affected by fomesafen: biosynthesis of amino acids, arginine biosynthesis, and 2-oxocarboxylic acid metabolism. CK vs RY and SY vs RY share a common differential pathway: degradation of flavonoids. Therefore, degradation of flavonoids may be a specific pathway contributing to the resistance observed in the RY group. Further analysis using Support Vector Machine (SVM) revealed that 4-hydroxy-2-oxoglutaric acid, citrulline, and L-ornithine are key metabolites involved in conferring resistance of A. retroflexus after fomesafen treatment.