Arbuscular mycorrhizal (AM) fungi form an obligate symbiosis with many wild and cultivated plants. The plant may benefit from improved nutrient uptake (particularly phosphorus) and resistance to drought, pests and disease. Compared to more natural habitats, arable systems support reduced diversity of AM fungi, with community structure shifted in favour of ruderal types. Physical disturbance is an important driver of these differences and reduced tillage systems might facilitate greater utilisation of the AM symbiosis as part of more sustainable production systems. In this study, the structure of root-associated AM fungal communities in barley grown under a range of tillage regimes was characterised, using high-throughput molecular methodology. AM fungal community structure was significantly influenced by tillage intensity, with soils subject to high tillage burden richest in ruderal types. These findings were consistent in both winter and spring barley cultivars. Significant temporal changes in AM fungal community structure suggested an important role for plant growth stage in determining AM fungal community dynamics. Functional differences can occur between fungal species and different fungus-plant combinations, and in the second part of this study the functional consequences of changes in community structure due to tillage were tested in a model system using intact field-soil cores. There were clear differences in AM fungal community structure due to differential physical disturbance. Although no functional effects were observed, this may have been due to the inherent limitations of recreating field conditions in microcosm experiments. The results of this comprehensive assessment of fine-scale spatial variation in AM fungal community structure in an arable system identify tillage as an important driver of AM fungal community dynamics, and plant growth stage is also a key factor which has rarely been addressed. Elucidating the functional significance of these changes remains essential in order to justify future changes to arable management practices.