In the context of climate change, the water-use efficiency (WUE) of highly productive tree varieties, such as eucalypts, has become a major issue for breeding programmes. This study set out to dissect the genetic architecture of carbon isotope composition (C-13), a proxy of WUE, across several environments. A family of Eucalyptus urophyllaxE.grandis was planted in three trials and phenotyped for C-13 and growth traits. High-resolution genetic maps enabled us to target genomic regions underlying C-13 quantitative trait loci (QTLs) on the E.grandis genome. Of the 15 QTLs identified for C-13, nine were stable across the environments and three displayed significant QTL-by-environment interaction, suggesting medium to high genetic determinism for this trait. Only one colocalization was found between growth and C-13. Gene ontology (GO) term enrichment analysis suggested candidate genes related to foliar C-13, including two involved in the regulation of stomatal movements. This study provides the first report of the genetic architecture of C-13 and its relation to growth in Eucalyptus. The low correlations found between the two traits at phenotypic and genetic levels suggest the possibility of improving the WUE of Eucalyptus varieties without having an impact on breeding for growth.