Aims - Understanding the belowground interactions in multi-species intercropping agroecosystems is critical to improving the cropping system's sustainability. This study aims to assess the sugarcane root colonization of the soil in plant crops when intercropped with legumes or spontaneous weeds in the inter-row. Methods - Roots were studied in the setting of three inter-row conditions: sugarcane with full chemical weed control, sugarcane-jack bean (Canavalia ensiformis) intercropping, and sugarcane with spontaneous weeds in the inter-row. Root mappings were conducted at 3.5, 6, 8, and 11 months after sugarcane planting in two vertical trenches per age and experimental condition (width 1.5 m x depth 1.2 m). Fine root distribution, root length densities, and a soil colonization index were estimated from the root intersects, in relation to plant root co-occurrence. Results - We found that both legumes and weeds in the inter-row decreased the total sugarcane fine root length by 17 to 30% compared to situation of chemical weed control from 6 months onwards. From 3.5 months, legumes or weeds impacted vertical and horizontal sugarcane root distribution, mainly in the topsoil. Consequently, the soil volume colonized by sugarcane roots decreased by 27% in both inter-row conditions throughout the crop cycle. This decrease was due to changes in root distribution and the co-occurrence of multi-species plant roots but not to changes in total root length. Conclusions - Our study pointed out the need to consider how the presence of other plant roots affects sugarcane root extension to understand and simulate the competition processes influencing intercropping performances.