Background and aims: We investigated the genetic diversity of arbuscular mycorrhizal fungi (AMF) in soils and the roots of Phalaris aquatica L., Trifolium subterraneum L., and Hordeum leporinum Link growing in limed and unlimed soil, the influence of lime application on AMF colonization and the relationship between AMF diversity and soil chemical properties. Methods: The sampling was conducted on a long-term liming experimental site, established in 1992, in which lime was applied every 6 years to maintain soil pH (in CaCl 2) at 5.5 in the 0-10 cm soil depth. Polymerase chain reaction, cloning and sequencing techniques were used to investigate the diversity of AMF. Results: Altogether, 438 AMF sequences from a total of 480 clones were obtained. Sequences of phylotypes Aca/Scu were detected exclusively in soil, while Glomus sp. (GlGr Ab) and an uncultured Glomus (UnGlGr A) were detected only in plant roots. Glomus mosseae (GlGr Aa) was the dominant AMF in the pastures examined; however, the proportion of G. mosseae was negatively correlated with soil pH, exchangeable Ca and available P. Generally, diversity of the AMF phylotypes was greater in the bulk unlimed soil and plants from this treatment when compared to the limed treatments. Conclusions: Long-term lime application changed soil nutrient availability and increased AMF colonization, but decreased AMF phylotype diversity, implying that soil chemistry may determine the distribution of AMF in acid soils. Future studies are required to explore the functions of these AMF groups and select the most efficient AMF for sustainable farming in acid soils.