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The use of resistant rootstocks is an inevitable trend in the development and production of grapes (Vitis sp.). The present study analyzed differences in the metabolites in grape seeds of different rootstock combinations (1103P, 5C, SO4, 3309C, 140R, and control) grafted onto ‘Cabernet Sauvignon’ (CS) wine grape (Vitis vinifera) scions (control, CS/CS, self-rooted grafting vines) using liquid chromatography–mass spectrometry (LC-MS) and nontargeted metabolomic techniques. Principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and orthogonal-partial least squares discriminant analysis identified 30 significant metabolites and 22 metabolic pathways in the seeds of CS that differed significantly from the control seeds. This study revealed that rootstocks influence metabolite concentrations and metabolic pathways (alanine–aspartate–glutamate pathway, arginine-proline pathway, and the tricarboxylic acid cycle) in the scion onto which they are grafted. The rootstocks increased the concentration of delphinidin-3-(6-acetylglucoside), peonidin 3-(6-p-coumarylglucoside), L-threonine, and D-tartaric in CS seeds. Appropriate rootstock combinations can be used to improve the quality of grape seeds by changing the concentrations of amino acids, organic acids, polyphenols, and vitamin B. This study provides a theoretical basis for selecting grape rootstocks and provides important insights for improving the quality of commercial products derived from grape seeds.