Plant growth, root characteristics and the stable carbon (δ13C) and oxygen (δ18O) composition were studied in durum wheat. Four recombinant inbred lines with good agronomic adaptation were grown under well watered (WW) and water stress (WS) conditions until mid-grain filling in lysimeters. Gas exchange was measured in the flag leaf just before harvest and then the aerial dry matter (Aerial DM), root weight density (RWD) and root length density (RLD) and the specific root length (SRL) were evaluated and the δ13C and δ18O of the roots, the flag leaf blade and the spike were analysed. Water stress decreased stomatal conductance, plant accumulated transpiration and Aerial DM, whereas δ13C and δ18O increased. Genotypic differences were found for all gas-exchange and root traits and isotope signatures. Aerial DM was positively correlated with RLD, regardless of the water regime, whereas it was negatively correlated with δ13C and δ18O, but only under WW conditions. Moreover, RWD and RLD were negatively related to both δ13C and δ18O under the WW regime, but no clear pattern existed under WS. Our study supports the use of δ13C and δ18O as proxies for selecting root traits associated with higher growth in the absence of water stress.