Evidence shows that tillage modifies soil properties, especially phosphorus (P) dynamics. Our objective was to disentangle long-term effects of P-fertilization and tillage on arbuscular mycorrhizal fungal (AMF) proliferation and community structure. Changes in the community structure of AMF and in the density of their hyphae and spores induced by moldboard plow (MP) or no till (NT), and fertilization with 0, 17.5, or 35 kg P ha(-1) were sought in the 0-15 cm and 15-30 cm soil layers after soybean harvest, at a long-term (17 years) experimental site in a humid continental zone of eastern Canada. The relationships among AMF, soil and plant attributes were examined. The 0-15 cm and 15-30 cm soil layers had different properties under NT, but were similar under MP, after 17 years, and MP increased soil available P levels. Phosphorus fertilization increased P levels in soil and in soybean. Treatment effects on AMF spore and hyphal density at 0-15 cm were greater than that at 15-30 cm, whereas effects on AMF community structure did not change with soil depths. At 0-15 cm, P-fertilization increased AMF spore density and reduced AMF hyphal density, and MP reduced AMF spore density. A total of eight AMF phylotypes were detected. Phosphorus fertilization reduced AMF phylotype richness and Shannon diversity index. Soil P availability increased under MP and hence the influence of P-fertilization treatments on the frequency of AMF phylotype detection varied with tillage system; it declined with P-fertilization under MP, but increased under NT. Phosphorus fertilization shifts resource partitioning in AMF propagules rather than in their hyphae, and degrades the genetic diversity of AMF in soil; tillage increases soil P availability and hence aggravates the impact of P-fertilization.