Proton NMR is one of the key analytical technologies in the field of metabolomics, as it allows one to combine untargeted, targeted, and quantitative metabolite measurements. One of NMR's greatest strengths is the ability to unambiguously identify compounds when present at mg/L concentrations, without the use of expensive or hard-to-source reference compounds. Furthermore, identification can be performed non-destructively on complex samples without the need for further sample preparation and isolation. Here, we describe a series of NMR experiments and data processing techniques to unambiguously identify the metabolite pyroglutamic acid (pGlu) in wine samples, without prior enrichment or separation from matrix compounds and other metabolites typically present in wine. Subsequently, the concentration of pGlu in 100 Australian wines was determined using standard NMR protocols. Statistical analysis demonstrated that occurrence of pGlu is associated with glutamic acid, is linked to vintage conditions and accumulated heat over the growing season, and is negatively associated with rainfall during the growing season. Overall, the results establish the presence and typical concentrations of the amino acid metabolite pGlu in Australian wine.