Downy mildew caused by Pseudoperonospora cubensis is a major disease of cucurbits worldwide. New genotypes of the pathogen have recently appeared in the USA, EU and Israel causing breakdown of genetic resistance, expansion of host range, and the appearance of a new A2 mating type. Seventy-eight P. cubensis isolates were collected during 1996–2011 from cucurbits fields in different regions of Turkey, Israel and the Czech Republic and genetic diversity was analysed using highly polymorphic ISSR and SRAP molecular markers. The data acquired showed remarkable genetic diversity within and among the isolates. While isolates from Turkey and Czech Republic exhibited uniform genetic background, the isolates from Israel were clearly distinguished from the others. The results may indicate on migration and/or frequent sexual reproduction of the pathogen in Israel. Moreover the selected markers can be suggested for monitoring genetic diversity within P. cubensis isolates in further studies.