Hybrid poplar clone NC–5339 (Populusalba × Populusgrandidentata cv. Crandon) was genetically modified for glyphosate (N-(phosphonomethyl)glycine) tolerance by Agrobacterium-mediated transformation with genetic constructs (pPMG 85/587 and pCGN 1107) that included the mutant aroA gene for 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19) and the neomycin phosphotransferase selectable marker gene. pCGN 1107 also harbored the coding sequence for a chloroplast transit peptide and the CaMV 35S promoter fused to the mutant aroA gene. Transformants were selected for kanamycin tolerance, and integration of the aroA gene was verified by Southern blot analysis. Cuttings of NC-5339 and the derived transformants were rooted and grown in glasshouses at separate locations, with maximum photosynthetic photon flux density of 1600 and 750 μmol•m−2•s−1. Productivity was assessed by growth studies and photosynthesis measurements at both locations. Glyphosate tolerance was tested by (i) measurement of chlorophyll concentration in herbicide-treated leaf discs and (ii) whole-plant spray tests. Plants transformed with construct pCGN 1107 were the most herbicide tolerant. Perhaps high-level expression of the aroA gene by the CaMV 35S promoter, transport of mutant EPSP synthase into the chloroplasts, or both facilitated glyphosate tolerance. Plants grown at higher photosynthetic photon flux densities (1600 vs. 750 μmol•m−2•s−1) had significantly higher maximum net photosynthesis (19.8 vs. 16.2 μmol•m−2•s−1) and more biomass accumulation (47.6 vs. 33.7 g). However, there were no significant differences between NC-5339 and transformants within location for net photosynthesis or any growth parameter. Genetic modification of hybrid poplar NC-5339 for glyphosate tolerance did not adversely affect plant productivity at either location.