This dataset consists of 35 rhizobox images. The experimental set up and image analysis pipeline have been described in the following paper: Alonso-Crespo IM, Weidlich EWA, Temperton VM, Delory BM (2022). Assembly history modulates vertical root distribution in a grassland experiment, Oikos, e08886. Root images were acquired using a digital camera (Canon EOS 5D Mark III) equipped with a 28 mm lens (Canon EF 28mm f/2.8) and connected to a computer. In addition to the camera and its associated holder, our image acquisition system consists of a metallic frame holding the rhizobox vertically and two LED tubes (4300 K, 60 cm length) positioned laterally (raking lighting) to provide uniform lighting conditions over the entire height of a rhizobox. The camera, the camera holder, the LED tubes, and the frame holding the rhizobox were installed inside a closed box whose internal walls were entirely covered with dark fabric. The camera faced each rhizobox directly and the camera lens was located 54 cm from the surface of the transparent front window. Images (3,840×5,760 pixels, width×height; resolution: 94.8 pixels/cm or ~240 dpi) were acquired using our camera’s remote live view and shooting option before being saved on a computer.