The Dutch elm disease pathogen Ophiostomanovo-ulmi is separated into subspecies novo-ulmi, formerly known as the Eurasian (EAN) race, and subspecies americana, formerly known as the North American (NAN) race. Both subspecies occur in Europe, and hybrids between them are suspected to have emerged in parts of Europe where their ranges overlap. Authenticated isolates of both subspecies were examined in order to determine whether fixed mutations occur in the cerato-ulmin (cu) and in the colony type gene (col1). One and six mutations were detected between subspp. novo-ulmi and americana in cu and col1, respectively. The mutation in cu and one mutation in col1 proved to be located within restriction sites, and were used for PCR-RFLP. This method provides a quick and reliable diagnostic method to differentiate the two subspecies. Seven isolates of O.novo-ulmi from Austria were suspected to be hybrids between subspp. novo-ulmi and americana, and were tested by PCR-RFLP of their cu and col1 genes. Two of the suspected hybrids from Austria (isolates AT73 and AT146) had the cu PCR-RFLP profile of ssp. americana and the col1 PCR-RFLP profile of ssp. novo-ulmi. Furthermore, a ssp. novo-ulmi tester isolate from Poland (P150) showed a similar hybrid pattern. This is the first evidence revealing recombination between two genes and thus hybridization between the two subspecies of O. novo-ulmi in nature.