BACKGROUND X-ray computed tomography (CT) has become a powerful tool for root phenotyping. Compared to rather classical, destructive methods, CT encompasses various advantages. In pot experiments the growth and development of the same individual root can be followed over time and in addition the unaltered configuration of the 3D root system architecture (RSA) interacting with a real field soil matrix can be studied. Yet, the throughput, which is essential for a more widespread application of CT for basic research or breeding programs, suffers from the bottleneck of rapid and standardized segmentation methods to extract root structures. Using available methods, root segmentation is done to a large extent manually, as it requires a lot of interactive parameter optimization and interpretation and therefore needs a lot of time. RESULTS Based on commercially available software, this paper presents a protocol that is faster, more standardized and more versatile compared to existing segmentation methods, particularly if used to analyse field samples collected in situ. To the knowledge of the authors this is the first study approaching to develop a comprehensive segmentation method suitable for comparatively large columns sampled in situ which contain complex, not necessarily connected root systems from multiple plants grown in undisturbed field soil. Root systems from several crops were sampled in situ and CT-volumes determined with the presented method were compared to root dry matter of washed root samples. A highly significant (P < 0.01) and strong correlation (R(2) = 0.84) was found, demonstrating the value of the presented method in the context of field research. Subsequent to segmentation, a method for the measurement of root thickness distribution has been used. Root thickness is a central RSA trait for various physiological research questions such as root growth in compacted soil or under oxygen deficient soil conditions, but hardly assessable in high throughput until today, due to a lack of available protocols. CONCLUSIONS Application of the presented protocol helps to overcome the segmentation bottleneck and can be considered a step forward to high throughput root phenotyping facilitating appropriate sample sizes desired by science and breeding.