Aims: Roots are vital organs for plants, but the assessment of root traits is difficult, particularly in deep soil layers under natural field conditions. A popular technique to investigate root growth under field or semi-field conditions is the use of minirhizotrons. However, the subsequent manual quantification process is time-consuming and prone to error. Methods: We developed a multispectral minirhizotron imaging system and a subsequent image analysis strategy for automated root detection. Five wavelengths in the visible (VIS) and near-infrared (NIR) spectrum are used to enhance living roots by a multivariate grouping of pixels based on differences in reflectance; background noise is suppressed by a vesselness enhancement filter. The system was tested against manual analysis of grid intersections for both spring barley (Hordeum vulgare L.) and perennial ryegrass (Lolium perenne L.) cultivars at two time-points. The images of living roots were captured in wet subsoil conditions with dead roots present from a previous crop. Results: Under the soil conditions used in the study, NIR reflectance (940 nm), provided limited ability to separate between rhizosphere components, compared to reflectance in the violet and blue light spectrum (405 nm and 450 nm). Multivariate image analysis of the spectral data, combined with vesselness enhancement and thresholding allowed for automated detection of living roots. Automated image analysis largely replicated the root intensity found during manual grid intersect analysis of the same images. Although some misclassification occurred, caused by elongated structures of dew and chalkstone with similar reflectance pattern as living root, the system provided similar or in some cases improved detection of genotypic differences in the total root length within each tube. Conclusion: The multispectral imaging system allows for automated detection of living roots in minirhizotron studies. The system requires considerably less time than traditional manual recording using grid intersections. The flexible training strategy used for root segmentation offers hope for the transfer to other rhizosphere components and other soil types of interest.