A significant proportion of arable land in south-western Australia is highly susceptible to subsoil compaction, which limits access of roots of wheat to water and nutrients at depth. Genotypic variation in the ability of roots to penetrate a hardpan has been reported for other cereals, using a pot technique, where a thin wax-layer of paraffin wax and petroleum jelly is placed in a soil column to simulate a hardpan. Previously we have modified and validated this technique for measuring root penetration ability of wheat seedlings under contrasting water regimes. Here we report on a series of five experiments (runs), two in well-watered and three in drought stress conditions, which evaluated seminal and nodal root penetration ability through thin wax layers among 24 Australian wheat cultivars and breeding lines (entries). These results were compared with observations on their rooting depths in two contrasting soil types in field trials, including a sandy duplex that contained a hardpan and a red clay that increased in soil strength with depth. Nodal roots ceased growth early under soil water deficit, and water uptake was instead dependant on seminal roots under conditions imposed in the pots. Plants were then reliant on the ability of seminal roots to penetrate the wax layer. Eight entries had superior root penetration ability in both well-watered and drought stressed conditions. Roots of three other entries, which failed to penetrate the wax layers, died under drought stress conditions. In field trials, there was a significant interaction between site and entry for maximum root depth. Our results from the pot studies and field trials indicate that there exists genotypic variation in root traits that are required to penetrate uniformly hard soil, dry soil or soil containing a hardpan. As four of the eight superior entries also showed superior root penetration ability at both sites in the field, there was an overall consistency, but there were exceptions at individual field sites. Factors likely to result in such exceptions were discussed, and topics for further research identified.