Arbuscular Mycorrhizal Fungi (AMF) form symbiotic associations with plant roots and can help mobilize nutrients from soil to the plant. The current study hypothesized that agroforestry systems of Rwanda harbor AMF with the potential to colonize roots of crops and hence enhance productivity. AMF spores were extracted from soil samples collected around most dominant tree species in Bugesera and Rubavu districts, respectively, representing semi-arid and sub-humid agro-ecological zones of Rwanda. The spores were morphologically identified and trap cultures for the most three predominant AMF spore morphotypes were made. This was followed by in-situ inoculation of maize with the fresh inocula from the trap cultures. Four different AMF genera were detected; Glomus, Gigaspora, Scutellospora and Acaulospora. All genera were found in both agroecological zones and in soil samples from all the host tree species with Glomus being the predominant group. All the maize inoculated with AMF had their roots colonized and Gigaspora performed best. The mean percentage root colonization varied between 40 and 70%. The study showed that soils under agroforestry systems of Rwanda harbor AMF with capability to colonize maize roots. These findings could be exploited in a view of selecting and developing well performing and adapted inocula to be used as bio-fertilizer.