As a unique feature, honey from the New Zealand manuka tree (Leptospermum scoparium) Contains substantial amounts of dihydroxyacetone (DHA) and methylglyoxal (MGO). Although MGO is a reactive intermediate in the Mallard reaction, very little is known about reactions of MGO with honey proteins. We hypothesized that the abundanee of MGO should result in a particular pattern of protein-bound Maillard reaction products (MRPs) in manuka honey. A protein-rich high molecular-weight fraction was isolated from 12 manuka and 8 non-manuka honeys and hydrolyzed enzymatically. By HPLC-MS/ MS, 8 MRPs, namely, N-e-fructosyllysine, N-8-maltulosyllysine, carboxymethyllysine, carboxyethyllysine (CEL), pyrraline, formyline, maltosine, and methylglyoxal-derived hydroimidazolone 1 (MG-H1), were quantitated. Compared to non-manuka honeys, the manuka honeys were characterized by high concentrations of CEL and MG-H1, whereas the formation of N-sfructosyllysine was suppressed, indicating concurrence reactions of glucose and MGO at the 8-amino group of protein-bound lysine. Up to 31% of the lysine and 8% of the arginine residues, respectively, in the manuka honey protein can be modified to CEL and MG-H1, respectively. CEL and MG-Hi concentrations correlated strongly with the MGO concentration of the honeys. Manuka honey possesses a special pattern of protein-bound MRPs, which might be used to prove the reliability of labeled MGO levels in honeys and possibly enable the detection of fraudulent MGO or DHA addition to honey.