The effect of elevated CO2 and nitrogen fertilization on the molecular chemistry of litter of three Populus species and associated soil organic matter (SOM) was investigated by pyrolysis-gas chromatography/mass spectrometry. The results are based on 147 quantified organic compounds in 24 litter samples. Litter of P. euramerica was clearly different from that of P. nigra and P. alba. The latter two had higher contents of proteins, polysaccharides, and cutin/cutan, while the former had higher contents of phenols and benzofurans/pyrans. The difference between replications was at least as large as the effect of treatments, so that no systematic chemical changes were attributable to CO2 effect or N-fertilization effect. The chemistry of SOM under the various species and treatments did not show significant changes either. The low number of available replicates that is two was clearly insufficient to overcome the effect of spatial variation on litter chemistry and detect small differences in molecular litter chemistry.