A system has been designed to monitor indirectly the spread of vesicular-arbuscular mycorrhizal fungal hyphae by periodically examining colonization of ‘trap’ or ‘receiver’ plants separated from inoculated ‘spreader’ plants by a root-free soil region. This system was used to monitor fungal hyphae spread from pre-inoculated clover plants through a 2 cm section containing substrates of various pH values, organic matter contents, nutrient availabilities, and water holding capacities into a section containing non-inoculated clover plants. After 5 weeks, fungal hyphae had colonized receiver plant roots through three of the six substrates. After 10 weeks receiver plant roots were colonized through all substrates but calcined clay. The system functioned well with no cross-contamination and is easily modified for studying other aspects of fungal hyphae growth.