Cryphonectria parasitica (Murr.)Barr, the ascomycete fungus that causes chestnut blight, is mainly responsible for the decline of the European chestnut (Castanea sativa Mill.) in Croatia. Since recovery of the chestnut forests is dependent on the presence of hypovirulent C. parasitica strains, we determined the incidence of hypovirulence in the central part of the country. Seventy eight isolates of the chestnut blight fungus C. parasitica were sampled from 2004 to 2006, from four populations (Markusevac 1, Markusevac 2, Samobor, Sljeme). Thirty four of them had white morphology, 19 intermediate, and 25 orange. Twenty six isolates (21 white, 2 intermediate and 3 orange) were screened for the presence of double stranded RNA (dsRNA) by cellulose chromatography and electrophoresis on agarose gels. Hypoviral dsRNA of approximately 12.7 kb in length was detected in all the white isolates tested. In 4 isolates smaller M-dsRNA was also detected. The identity of the hypoviral isolates was determined by reverse transcription, polymerase chain reaction of two hypoviral open reading frames and restriction fragment length polymorphism (RFLP) analysis. Restriction patterns of the amplified products showed little variation among the examined hypoviruses from Croatia. Six patterns were completely identical with that of a hypoviral isolate collected in Croatia 22 years ago, which suggest a slow evolution of the hypovirus.