This study evaluated the potential of transgenic Populus trichocarpa with antisense 4CL for reduced total lignin and sense Cald5H for increased S/G ratio in a short rotation woody cropping (SRWC) system for bioethanol production in the Southeast USA. Trees produced from tissue-culture were planted in the Coastal Plain, Piedmont, and Mountain regions of North Carolina, USA. Trees were observed for growth differences and biomass recorded for two coppices. Insoluble lignin and S/G ratio were determined by molecular beam mass spectroscopy after the second coppice. Survival, growth form, and biomass were very consistent within construct lines. Higher total lignin content and S/G ratio were positively correlated with total aboveground biomass. The low-lignin phenotype was not completely maintained in the field, with total lignin content increasing on average more than 30.0% at all sites by the second coppice The capacity to upregulate lignin in the event of environmental stress may have helped some low-lignin lines to survive. More research focused on promising construct lines in appropriate environmental conditions is needed to clarify if a significant reduction in lignin can be achieved on a plantation scale, and whether that reduction will translate into increased efficiency of enzymatic hydrolysis. © 2014 Elsevier Ltd.