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Typically, root system architecture (RSA) is not visible, and realistically, high-throughput methods for RSA trait phenotyping should capture key features of developing root systems in solid substrates in 3D. In a published 2-D study using thin rhizoboxes, vermiculite as a growing medium, and photography for imaging, triplicates of 137 soybean cultivars were phenotyped for their RSA. In the transition to 3-D work using X-ray computed tomography (CT) scanning and mineral soil, two research questions are addressed: (1) how different is the soybean RSA characterization between the two phenotyping systems; and (2) is a direct comparison of the results reliable? Prior to a full-scale study in 3D, we grew, in pots filled with sand, triplicates of the Casino and OAC Woodstock cultivars that had shown the most contrasting RSAs in the 2-D study, and CT scanned them at the V1 vegetative stage of development of the shoots. Differences between soybean cultivars in RSA traits, such as total root length and fractal dimension (FD), observed in 2D, can change in 3D. In particular, in 2D, the mean FD values are 1.48 ± 0.16 (OAC Woodstock) vs. 1.31 ± 0.16 (Casino), whereas in 3D, they are 1.52 ± 0.14 (OAC Woodstock) vs. 1.24 ± 0.13 (Casino), indicating variations in RSA complexity.