Although a large number of AroA enzymes (5-enopyruvylshikimate-3-phosphate synthase [EPSPS]) have been identified, cloned and tested for glyphosate resistance, only AroA variants derived from Agrobacterium tumefaciens strain CP4 have been successfully used commercially. We have now used a polymerase chain reaction (PCR)-based two-step DNA synthesis (PTDS) method to synthesize an aroA gene (aroA (H. orenii) ) from Halothermothrix orenii H168 encoding a new EPSPS similar to AroA (A. tumefaciens CP4.) AroA (H. orenii) was then expressed in Escherichia coli and key kinetic values of the purified enzyme were determined. Kinetic analysis of AroA (H. orenii) indicated that the full-length enzyme exhibited increased tolerance to glyphosate compared with E. coli AroA (E. coli) while retaining a high affinity for the substrate phosphoenolpyruvate. Transgenic Arabidopsis plants containing aroA (H. orenii) were resistant to 15 mM glyphosate. Site-directed mutagenesis showed that residues Thr355Ser affected the affinity of AroA (H. orenii) for glyphosate, providing further evidence that specific amino acid residues are responsible for differences in enzymatic behavior among different AroA enzymes.