In vegetated terrestrial ecosystems, carbon in below- and aboveground biomass (BGB, AGB) often constitutes a significant component of total-ecosystem carbon stock. Because carbon in the BGB is difficult to measure, it is often estimated using BGB to AGB ratios. However, this ratio can change markedly along resource gradients, such as water availability, which can lead to substantial errors in BGB estimates. In this study, BGB and AGB sampling was carried out in Eucalyptus populnea-dominated woodland communities of northeast Australia to examine patterns of BGB to AGB ratio and vertical root distribution at three sites along a rainfall gradient (367, 602, and 1,101 mm). At each site, a vegetation inventory was undertaken on five transects (100 × 4 m), and trees representing the E. populnea vegetation structure were harvested and excavated to measure aboveground and coarse-root (diameter of at least 15 mm) biomass. Biomass of fine and small roots (diameter less than 15 mm) at each site was estimated from 40 cores sampled to 1 m depth. The BGB to AGB ratio of E. populnea-dominated woodland plant communities declined from 0.58 at the xeric end to 0.36 at the mesic end of the rainfall gradient. This was due to a marked decline in AGB with increased aridity whereas the BGB was relatively stable. The vertical distribution of fine roots in the top 1 m of soil varied along the rainfall gradient. The mesic sites had more fine-root biomass (FRB) in the upper soil profile and less at depth than the xeric site. Accordingly, at the xeric site, a much larger proportion of FRB was found at depth compared to the mesic sites. The vertical distribution patterns of small roots of the E. populnea woodland plant communities were consistently )-shaped, with the highest biomass occurring at 15–30-cm depth. The potential significance of such a rooting pattern for grass–tree and shrub–tree co-existence in these ecosystems is discussed. Overall, our results revealed marked changes in BGB to AGB ratio of E. populnea woodland communities along a rainfall gradient. Because E. populnea woodlands cover a large area (96 M ha), their contribution to continental-scale carbon sequestration and greenhouse gas emission can be substantial. Use of the rainfall-zone-specific ratios found in this study, in lieu of a single generic ratio for the entire region, will significantly improve estimates of BGB carbon stocks in these woodlands. In the absence of more specific data, our results will also be relevant in other regions with similar vegetation and rainfall gradients (that is, arid and semiarid woodland ecosystems).