One hundred and six isolates of the Dutch elm disease (DED) fungi Ophiostoma ulmi and Ophiostoma novo-ulmi were collected from elm trees with symptoms in 15 regions of Spain. Isolates were compared with eight reference strains belonging to O. ulmi and the two subspecies of O. novo-ulmi. The purpose of this study was to assign Spanish isolates to species and subspecies of the DED fungi and to analyse the genetic variability within the Spanish populations of these pathogens. Isolates were examined for their growth rates, colony morphologies and fertility responses and by using random amplified polymorphic DNA (RAPD) markers. Six isolates were identified as O. ulmi, 16 as O. novo-ulmi ssp. novo-ulmi and 78 as O. novo-ulmi ssp. americana. Nei's and Shannon's diversity indices and the upgma dendrogram from RAPD profiles indicated a high level of variation among isolates, probably reflecting post-epidemic status of DED in Spain. Although most isolates were separated into three major clusters representing the three taxa of DED fungi in the RAPD analysis, two isolates from central Spain clustered between O. novo-ulmi ssp. americana and O. novo-ulmi ssp. novo-ulmi, and four isolates from Mallorca clustered between O. ulmi and the group representing O. novo-ulmi ssp. novo-ulmi. Mating tests conducted with these isolates revealed a variety of fertility responses. The novel combinations between molecular profile and fertility reaction suggest that three isolates from Mallorca may be interspecific hybrids of the DED fungi.