Ensuring food security through sustainable practices while reducing greenhouse gas emissions are key challenges in modern agriculture. Utilising genetic variability within a crop species to identify varieties with higher root biomass carbon (C) could help address these challenges. It is thus crucial to quantify and understand intra-specific above- and belowground performance under varying environmental conditions. The study objectives were to: (a) quantify root biomass and depth distribution in different winter wheat varieties under various pedoclimatic conditions, (b) investigate the influence of variety and pedoclimatic conditions on the relationship between above- and belowground biomass production, and (c) assess whether optimised winter wheat variety selection can lead to both greater root biomass C and yield, boosting C accrual. Root biomass, root distribution to 1 m soil depth and root-to-shoot ratios were assessed in 10 different winter wheat varieties grown at 11 experimental sites covering a European climatic gradient from Spain to Norway. Median root biomass down to 1 m depth was 1.4 ± 0.7 Mg ha−1. The primary explanatory factor was site, accounting for 60% of the variation in root biomass production, while the genetic diversity between wheat varieties explained 9.5%. Precipitation had a significantly negative effect on total root biomass, especially in subsoil. Significant differences were also observed between varieties in root-to-shoot ratios and grain yield. The difference between the variety with the lowest root biomass and the one with the highest across sites was on average 0.9 Mg ha−1 which is an increase of 45%. Pedoclimatic conditions had a greater influence than variety, and determined the relationship's direction between root biomass and grain yield. A site-specific approach is, therefore, needed to realise the full potential for increased root biomass and yield offered by optimised variety selection.