Pyrosequencing analysis was performed on soils from Italian chestnut groves to evaluate the diversity of the resident Phytophthora community. Sequences analysed with a custom-database discriminated 15 pathogenic Phytophthoras including species common to chestnut soils, while a total of 9 species were detected with baiting. The two sites studied differed in Phytophthora diversity and the presence of specific taxa responded to specific ecological traits of the sites. Furthermore, some species not previously recorded were represented by a discrete number of reads; among these species, P. ramorum was detected at both sites. Pyrosequencing demonstrated to be a very sensitive technique to describe Phytophthora community in soil, able to detect species not easy to be isolated from soil with standard baiting techniques. In particular, pyrosequencing is an highly efficient tool for investigating the colonisation of new environments by alien species, and for ecological and adaptive studies coupled to biological detection methods. This study represents the first application of pyrosequencing for describing Phytophthoras in environmental soil samples. This article is protected by copyright. All rights reserved.