Herbicide resistance is the most widely used transgenic crop trait for broad-spectrum control of weeds. Here we report a novel 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene (Gr5aroA) isolated from glyphosate-contaminated soil. The full Gr5aroA gene was 1,819 bp and contained a 1,341-bp open reading frame encoding a 47-kDa protein. Phylogenetic analysis showed that Gr5aroA is a class I EPSPS even though most such enzymes are naturally sensitive to glyphosate. Interestingly, Gr5aroA protein contained highly conserved PEP and S3P binding residues (Glu-351) and several motifs insensitive to glyphosate. Transgenic Gr5aroA plants (T0) grew normally and produced seeds which we treated with a high-glyphosate solution (4× recommended spray). Analysis of the T1 progenies showed that Gr5aroA was inherited at a Mendelian 3:1 segregation ratios and that glyphosate tolerance in T1 plants was unchanged. Our results show the Gr5aroA gene to be a promising candidate for the development of commercial transgenic crops with high glyphosate tolerance. © 2013 Springer Science+Business Media Dordrecht.