ABSTRACT: Mycorrhizal symbioses are considered indicators of ecosystem biodiversity. However, their diversity and relevance in arid and semi-arid ecosystems are poorly understood. This study addressed this subject, the main objective being to evaluate arbuscular mycorrhizal fungi (AMF) diversity and heterogeneity in a semi-arid region. Samples of bulk and rhizosphere soil and fine roots of Medicago truncatula were collected at four different sites with the same aridity index (6.1), in Bou-Hedma National Park, Tunisia, a semi-arid ecosystem. AMF taxa were assessed by 454- pyrosequencing and identified by BLAST matching of operational taxonomic units (OTUs) against the MaarjAM database, targeting AMF SSU rRNA gene diversity. Roots were the hotspots of AMF diversity (107 OTUs out of a total of 138). Of the 138 OTUs, 113 found correspondence in the MaarjAM database, with 32 AMF virtual taxa (VTX),19 Site-exclusive (SE) and 13 common to at least two sites (Non-site exclusive, NSE); the remaining 25 OTUs grouped in 16 putative new AMF taxa (pNTX), each one consisting of OTUs sharing pairwise distances not higher than 3%. We found a high diversity and heterogeneity of AMF across the four sites, which showed, in a regression analysis, significant relation to six out of the eight environmental parameters evaluated: grazing activity and soil texture, electrical conductivity, organic matter, total phosphorus and total nitrogen. AMF colonization of plants also presented significant differences among the four sites, as well as spore density, microbial biomass and several enzymatic activities (dehydrogenase, β-glucosidase and phosphatase) evaluated in rhizosphere soils. The four sites clustered in two groups in a hierarchical clustering evaluation based on their AMF diversity (total numbers of OTU, VTX and pNTX) and the parameters referred above. The crucial role of abiotic factors, other than aridity index, on AMF community composition, was evidenced by the high heterogeneity found between AMF communities across sites under identical aridity conditions.