Soil characteristics may be an important factor structuring fungal communities. We relate the case of the ectomycorrhizal fungi (EMF) Pisolithus spp. according to changes in soil chemistry in a Quercus suber plantation located in the Maâmora forest (Morocco). Intrageneric variability of 100 fruit bodies was studied, using morphological characterization, Restriction Fragment Length Polymorphism (RFLP) analyses and Internal Transcribed Spacer (ITS) sequencing. Two Pisolithus spp. genotypes were identified: 97% of the fruit bodies represent Pisolithus arhizus, the remaining 3% correspond to P. species 4 as previously described by Martin and colleagues in 2002. Multivariate analysis (PCA) showed that species structure was strongly correlated with soil characteristics. P. arhizus fruit bodies were frequently found in the Eastern part of the plot at the low molasic clay, N and P contents, while P. species 4 were collected only in the Western part at the high molasic clay, N and P contents. To determine whether this change in fruit bodie structure was expressed at the belowground level, we used morphological and molecular techniques to characterize the Pisolithus ectomycorrhizas. Only ectomycorrhizas of P. arhizus were identified at the low P-N-molassic clay site and disappeared completely at the high P-N-red clay site, where no P. species 4 mycorrhizas have been found. Regression analysis showed that P. arhizus mycorrhizas structure was strongly correlated with soil characteristics. However, autocorrelation among soil parameters makes it difficult to isolate the effects of individual parameters. These results show the local-scale impact of natural spatial heterogeneity on an ECM fungal genus.