Background: Second generation (2G) bioenergy from lignocellulosic feedstocks has the potential to develop as a sustainable source of renewable energy; however, significant hurdles still remain for large-scale commercialisation. Populus is considered as a promising 2G feedstock and understanding the genetic basis of biomass yield and feedstock quality are a research priority in this model tree species. Results: We report the first coppiced biomass study for 714 members of a wide population of European black poplar (Populus nigra L.), a native European tree, selected from 20 river populations ranging in latitude and longitude between 40.5 and 52.1 degrees N and 1.0 and 16.4 degrees E, respectively. When grown at a single site in southern UK, significant Site of Origin (SO) effects were seen for 14 of the 15 directly measured or derived traits including biomass yield, leaf area and stomatal index. There was significant correlation (p < 0.001) between biomass yield traits over 3 years of harvest which identified leaf size and cell production as strong predictors of biomass yield. A 12 K Illumina genotyping array (constructed from 10,331 SNPs in 14 QTL regions and 4648 genes) highlighted significant population genetic structure with pairwise F-ST showing strong differentiation (p < 0.001) between the Spanish and Italian subpopulations. Robust associations reaching genome-wide significance are reported for main stem height and cell number per leaf; two traits tightly linked to biomass yield. These genotyping and phenotypic data were also used to show the presence of significant isolation by distance (IBD) and isolation by adaption (IBA) within this population. Conclusions: The three associations identified reaching genome-wide significance at p < 0.05 include a transcription factor; a putative stress response gene and a gene of unknown function. None of them have been previously linked to bioenergy yield; were shown to be differentially expressed in a panel of three selected genotypes from the collection and represent exciting, novel candidates for further study in a bioenergy tree native to Europe and Euro-Asia. A further 26 markers (22 genes) were found to reach putative significance and are also of interest for biomass yield, leaf area, epidermal cell expansion and stomatal patterning. This research on European P. nigra provides an important foundation for the development of commercial native trees for bioenergy and for advanced, molecular breeding in these species.