Metabolic syndrome and type 2 diabetes are associated with increased risk of breast cancer development and progression. Methylglyoxal (MG), a glycolysis by-product, is generated through a non-enzymatic reaction from triose-phosphate intermediates. This dicarbonyl compound is highly reactive and contributes to the accumulation of advanced glycation end products. In this study, we analyzed the accumulation of Arg-pyrimidine, a MG-arginine adduct, in human breast adenocarcinoma and we observed a consistent increase of Arg-pyrimidine in cancer cells when compared with the non-tumoral counterpart. Further immunohistochemical comparative analysis of breast cancer subtypes revealed that triple negative lesions exhibited low accumulation of Arg-pyrimidine compared with other subtypes. Interestingly, the activity of glyoxalase 1 (Glo-1), an enzyme that detoxifies MG, was significantly higher in triple negative than in other subtype lesions, suggesting that these aggressive tumors are able to develop an efficient response against dicarbonyl stress. Using breast cancer cell lines, we substantiated these clinical observations by showing that, in contrast to triple positive, triple negative cells induced Glo-1 expression and activity in response to MG treatment. This is the first report that Arg-pyrimidine adduct accumulation is a consistent event in human breast cancer with a differential detection between triple negative and other breast cancer subtypes.