Competition between crops and weeds may be stronger at the root than at the shoot level, but belowground competition remains poorly understood, due to the lack of suitable methods for root discrimination. Using a transgenic maize line expressing green fluorescent protein (GFP), we nondestructively discriminated maize roots from weed roots. Interactions between GFP-expressing maize, common lambsquarters, and redroot pigweed were studied in two different experiments with plants arranged in rows at a higher plant density (using boxes with a surface area of 0.09 m2) and in single-plant arrangements (using boxes with a surface area of 0.48 m2). Root density was screened using minirhizotrons. Relative to maize that was grown alone, maize root density was reduced from 41 to 87% when it was grown with redroot pigweed and from 27 to 73% when it was grown with common lambsquarters compared to maize grown alone. The calculated root : shoot ratios as well as the results of shoot dry weight and root density showed that both weed species restricted root growth more than they restricted shoot growth of maize. The effect of maize on the root density of the weeds ranged from a reduction of 25% to an increase of 23% for common lambsquarters and a reduction of 42 to 6% for redroot pigweed. This study constitutes the first direct quantification of root growth and distribution of maize growing together with weeds. Here we demonstrate that the innovative use of transgenic GFP-expressing maize combined with the minirhizotron technique offers new insights on the nature of the response of major crops to belowground competition with weeds.