RhizoVision Explorer is free and open-source software developed for estimating root traits from images acquired from a flatbed scanner or camera. Root images are expected to have a high contrast of roots with the background, homogenous lighting, and minimal overlapping of roots. The software is built in C++ for speed and stability, using QT for the graphical user interface and OpenCV for image processing. Traits extracted by RhizoVision Explorer have been extensively validated using a physical copper wire ground truth image set, thousands of simulated roots, and comparisons with other image analysis software. The default “Broken Roots” mode is meant for roots washed out from soil cores or pots that are disconnected and provides length, volume, branching frequency, among other features, with the ability to bin measurements based on diameter thresholds. The “Whole Root” mode extracts additional root system architecture features of more intact root systems, especially excavated root crowns or rhizoboxes, such as the convex hull, angles, and holes. The software supports multiple regions of interest, batch mode, and user-defined export of processed images with overlaid features for use in reports. Recommended scanners and plans for scanning trays Plans for RhizoVision Crown for camera-based root crown phenotyping A brief demo of the software is available on YouTube. Minimum requirements: Windows 10 or 8 operating system, an Intel or AMD x86_64 processor, and 8 GB of RAM. If the processor supports Intel AVX 2.0, the code is optimized. License: You can redistribute it and/or modify it as permissible under the GNU General Public License. Source code: The source code is available on GitHub. Citation: The software creators humbly request you cite the software and manuscript if used for data presented in presentations and publications. When citing the software, please use the DOI for the version you used for reproducibility. Seethepalli, A. and York, L. M. (2020). RhizoVision Explorer - Interactive software for generalized root image analysis designed for everyone (Version 2.0.3). Zenodo. http://doi.org/10.5281/zenodo.4095629 The manuscript that describes and validates RhizoVision Explorer can be cited as: Seethepalli, A., Dhakal, K., Griffiths, M., Guo, H., Freschet, G. T., York, L. M. (2021). RhizoVision Explorer: Open-source software for root image analysis and measurement standardization. AoB PLANTS, plab056, https://doi.org/10.1093/aobpla/plab056 Please consider sharing your datasets, statistical analysis code, and even the settings files used for RhizoVision Explorer in a publicly accessible repository, such as Zenodo, DataDryad, Figshare, CyVerse Data Commons, or other. Installing the program: Download RhizoVisionExplorer-2.0.2-windows-x64.zip below and uncompress the ZIP file to a local directory and run RhizoVisionExplorer.exe. You may be prompted to confirm the security of the application by Windows Defender or other applications. An illustrated manual is included in the manual folder of the RVE download as a PDF. The imageexamples folder includes PNG files of wheat root crowns taken with the RhizoVision Crown platform and JPG files of scanned disconnected wheat roots at 600 DPI for testing purposes. Please contact the creators with concerns or comments: Anand Seethepalli anand_seethepalli@yahoo.co.in Larry M. York yorklm@ornl.gov Brief change log below, see full log as 'changelog.txt' included with the download. V2.0.3 has no feature changes but reflects a change in copyright courtesy of Noble Research Institute, LLC to Anand Seethepalli and Larry York and updated the license to the GNU GPL from the modified Noble GPL. V2.0.2 was the first public release and had some new features. V2.0.1 was the closed access beta version used for wide testing.