The aim of this research were to investigate the genetic basis for variation in root penetration ability and associated traits in the mapping population derived from the Australian Bread wheat cultivars Halberd and Cranbrook in soil columns containing wax layers grown in controlled conditions and to compare this with performance in the field. Root and shoot traits of the DHL from a cross of Halberd and Cranbrook were evaluated in soil columns containing wax layers. Contrasting DHLs that varied in the ability to penetrate a wax layer in soil columns were then evaluated for maximum root depth in the field on contrasting soils at Merredin, Western Australia. Genetic control was complex and numerous QTL (53 in total) were located across most chromosomes that had a small genetic effect (LOD scores of 3.2 to 9.1). Of these QTL, 29 were associated with root traits, 37% of which were contributed positively by the Halberd with key traits being located on chromosomes 2D, 4A, 6B and 7B. Variation in root traits of DHL in soil columns was linked with field performance. Despite the complexity of the traits and a large number of small QTL, the results can be potentially used to explore allelic diversity in root traits for hardpan penetration.