Genotypic variation in nitrogen-use traits remains largely unexplored in trees on a range-wide scale, either in field studies or under controlled experiments. Understanding natural variation in nitrogen-related traits and their relationships to climate is essential for studying local adaptation and advancing breeding efforts. In this study, we took advantage of a large collection of black cottonwood genotypes covering a major portion of the species' natural range, to study the genetic variation in nitrogen isotope discrimination (Delta 15N). Nearly 350 unrelated wild genotypes were grown under steady-state hydroponic conditions and analyzed for growth and Delta 15N-related traits. Differences in biomass, root-to-shoot ratio, whole-plant and organ-level nitrogen percentages, and Delta 15N were found between genotypes and populations. Leaf nitrogen percentage and root-to-shoot ratio were significantly correlated to geographic and climatic variables, implying natural selection for lower leaf nitrogen and lower root-to-shoot ratio in regions with longer growing seasons and a lower risk of drought. Root Delta 15N and (less so) leaf Delta 15N correlated with geographic and climatic variables, and measurements from either tissue provide a reasonable indication of plant nitrogen uptake efficiency. A genome-wide association study was conducted on leaf and root Delta 15N, leaf nitrogen percentage and root-to-shoot ratio. The analysis identified a candidate gene encoding glutaminyl-tRNA synthetase linked to root Delta 15N, but found no significant associations with genes involved in nitrate transport or assimilation. However, multiple associations were detected for root-to-shoot ratio and leaf nitrogen percentage, both of which affect isotope-based calculations of root nitrogen efflux/influx and leaf nitrogen assimilation activity.