Roots take up most of the resources required by a plant, but a lack of efficient research tools hinders our understanding of the function and relevance of the root system. This is especially evident when the research focus is not on a single plant, but on multiple plants that share the same soil resources. None of the available methods allow for simple, inexpensive, non-destructive, and objective assignment of observed roots in a mixture of plants to a target plant. Here, we demonstrate that transgenic plants expressing the green fluorescent protein (GFP), combined with the well established minirhizotron technique, is a route to overcoming this limitation. We planted transgenic maize (Zea mays L.) in combination with either its corresponding wild type, Italian ryegrass (Lolium multiflorum Lam.), or soybean (Glycine max (L.) Merr.). Identification of fluorescent roots allows the relative distribution of roots of each plant type and their interaction and interference with each other to be observed. The selected plants are suitable for model experiments to unravel fundamental belowground ecological processes. Because genetic transformation of plants is an established technique that can be applied to a large set of plant species, this method will be of interest to a broad range of research areas.