During 1997–2000 the virulence variation and distribution of virulence phenotypes of Bremia lactucae (lettuce downy mildew) in natural populations of Lactuca serriola (prickly lettuce) were studied. Altogether 139 isolates of B. lactucae originating from the Czech Republic, France and Germany were examined for the presence of 27 virulence factors (v-factors) and their combinations. In the Czech population, 37 different v-phenotypes (P1–P37) of B. lactucae were found to occur on L. serriola. Most v-phenotypes were characterized by v-factors that match resistance (Dm genes/R-factors) carried by L. serriola. A wide diversity of v-phenotypes was recorded every year, but most were rare and did not reappear in other populations of B. lactucae. The three v-phenotypes P21, P28 and P29 were most frequent and widely distributed. There was variation both between spatially isolated populations and within populations of the pathogen. Geographic differences in virulence were found for the southern parts of Moravia, where the phenotypic composition of pathogen populations was completely different from the remaining part of the area investigated. However, some populations had v-phenotypes similar to those of spatially distant populations in Moravia.