The use of a permanent vegetation cover or frequent tillage in vineyards may affect soil water budget, nutrient availability, soil compaction, soil erosion and soil microbe biodiversity, and through all these and other factors also yield and wine quality parameters. The abundance and diversity of arbuscular mycorrhizal fungi (AMF) might also be influenced, but so far effects on AMF by permanent vegetation cover (= no-tillage systems) or repeated chiseling and rotary-tillaging have rarely been compared in vineyards. The objective of this on-farm study was to determine AMF species richness and diversity in two adjacent vineyards in Palatinate (SW Germany). In both vineyards, grown on fertile Luvisols, the var. “Pinot Gris” was grown for 39 years, but with different soil cultivation and different fertilization strategies. In one vineyard, soil was maintained periodically without vegetation by passing rotatory cultivator and chiseling between the grapevine rows (‘inter-rows’) several times per year, preferably during spring and summer and in dependency of rainfall and ‘weed’ growth, and fertilization was mainly by organic fertilizers in the last ten years before soil sampling. In the other vineyard, a permanent vegetation has been established since planting, dominated by Lolium perenne, and mineral fertilizers were exclusively applied. Despite of similar high nutrient availability in both soils, in particular of phosphorus, astonishing high AMF species richness and diversity were found in both vineyards. In the no-tillage inter-rows, 34 AMF species were found, with a species composition typically for Central European permanent grasslands (Shannon diversity index 2.45). In the tillage system 24 AMF species were found with a composition as known for extensively used, cultivated Central European croplands (diversity index 2.26). We conclude that above all soil cultivation has affected AMF diversity in these Central European vineyards, while the level and type of fertilization affected the AMF communities only on a minor level.