Methylglyoxal is a kind of poisonous metabolite that can react with RNA, DNA and protein, which generally results in a number of side advert effects to cell. Glyoxalase I is a member of glyoxalase system that can detoxify methylglyoxal. An EST encoding a glyoxalase I was isolated from a SSH (suppression subtractive hybridization)-cDNA library of wheat spike inoculated by Fusarium graminearum. The corresponding full length gene, named TaGly I, was cloned, sequenced and characterized. Its genomic sequence consists of 2,719 bp, including seven exons and six introns, and its coding sequence is 929 bp with an open reading frame encoding 291 amino acids. Sequence alignment showed that there were two glyoxalase I domains in the deduced protein sequence. By using specific primers, TaGly I was mapped to chromosome 7D of wheat via a set of durum wheat 'Langdon' D-genome disomic-substitution lines. The result of Real-time quantitative polymerase chain reaction demonstrated that TaGly I was induced by the inoculation of Fusarium graminearum in wheat spikes. Additionally, it was also induced by high concentration of NaCl and ZnCl2. When TaGly I was overexpressed in tobacco leaves via Agrobacterium tumefaciens infection, the transgenic tobacco showed stronger tolerance to ZnCl2 stress relative to transgenic control with GFP. The above facts indicated that TaGly I might play a role in response to diverse stresses in plants.