In the summer of 2009, high levels of mortality among white clawed crayfish Austropotamobius pallipes were observed in 3 watercourses of central Italy. PCR and culture methods were used to detect the causative agent of the disease. Two strains of Aphanomyces spp. were isolated and identified by PCR and DNA sequencing as Aphanomyces astaci and A. repetans. This is the first crayfish plague outbreak in Italy to be confirmed by the isolation in culture of a pathogen from Austropotamobius pallipes.