In September 2009, isolates of Phaeoacremonium sp., were isolated from the roots and rootstock of an adult grapevine and from the rootstock of a young plant. The scions of both plants belonged to cv. Tempranillo and the rootstocks were both of type 110R. Only the adult plants collected in the Ribera de Duero area, Castilla y Leon, Spain, showed external 'esca' disease symptoms (interveinal chlorotic leaves and internal black wood streaking). Morphological, molecular and pathogenicity tests identified the pathogens as P. viticola and P. hispanicum. This is thought to be the first report of P. viticola and P. hispanicum in Castilla y Leon, Spain.