This study compared the leaf transcription profiles, physiological characteristics and primary metabolites of two Populus tremuloides genotypes (clones 216 and 271) known to differ in their responses to long-term elevated [CO2] (e[CO2]) at the Aspen free-air CO2 enrichment site near Rhinelander, WI, USA. The physiological responses of these clones were similar in terms of photosynthesis, stomatal conductance and leaf area index under e[CO2], yet very different in terms of growth enhancement (0-10% in clone 216; 40-50% in clone 271). Although few genes responded to long-term exposure to e[CO2], the transcriptional activity of leaf e[CO2]-responsive genes was distinctly different between the clones, differentially impacting multiple pathways during both early and late growing seasons. An analysis of transcript abundance and carbon/nitrogen biochemistry suggested that the CO2-responsive clone (271) partitions carbon into pathways associated with active defense/response to stress, carbohydrate/starch biosynthesis and subsequent growth. The CO2-unresponsive clone (216) partitions carbon into pathways associated with passive defense (e.g. lignin, phenylpropanoid) and cell wall thickening. This study indicates that there is significant variation in expression patterns between different tree genotypes in response to long-term exposure to e[CO2]. Consequently, future efforts to improve productivity or other advantageous traits for carbon sequestration should include an examination of genetic variability in CO2 responsiveness.