Soil core samples were taken from a multi-species grassland experiment with field plots of monocultures and mixtures of six grassland plant species: Lolium perenne L. (PRG), Phleum pratense L. (TIM), Trifolium pratense L. (RC), Trifolium repens L. (WC), Cichorium intybus L. (CHIC), and Plantago lanceolata L.. The multi-species plots had a two species mixture with Trifolium repens L. and Lolium perenne L. (PRGWC), and a 6 species mixture with all species mentioned above. The cores were separated into soil depths of 0-10 cm, 10-15 cm and 15-20 cm and the roots separated from the soil. A ground-truth image set was created to simulate fine roots using fishing line. The fishing line used was a clear copolymer monofilament (GreysTM Greylon Tippet Material 3 lb), measured using a scanning electron microscope (Hitachi SU8200) to be 0.14 mm in diameter. The fishing line was used in its clear colour or coloured black using a permanent marker to simulate unstained and stained fine roots respectively. The fishing line was cut into lengths of 30 cm or 5 cm. Roots and fishing line were scanned using an Epson Perfection V800 flatbed scanner at 600 dpi. The Roots ZIP file contains a folder for the scanned root images and the Line zip file contains a folder with the scanned fishing line. The excel spreadsheet describes the naming convention for the images. Further details about the root sampling and image acquisition can be found in the publication that analyses these images: https://doi.org/10.1002/ppj2.20034