<p>Downy mildew disease caused by <i>Pseudoperonospora cubensis</i> is a major constraint of cucurbit production in India. Although <i>P. cubensis</i> is prevalent in all the cucurbit growing regions of Punjab, there is a lack of information about its diversity and pathogenicity to guide the development of effective management strategies. In total, 28 isolates of <i>P. cubensis</i> were obtained from cucumber and muskmelon fields in different agroclimatic zones of Punjab, with the following objectives to characterize their morphology, based on the length and breadth of sporangia, the total length of sporangiophore, the height of first branching, the width of the trunk, length of larger ultimate branchlet, length of smaller ultimate branchlet; and to determine the pathotypic variability of <i>P. cubensis</i> cucumber isolates on five artificially inoculated cucurbit hosts under controlled condition in greenhouse. We found considerable differences in morphological parameters like the size of sporangiophore and sporangia. There were also differences in the virulence spectrum of the pathogen. Six isolates of <i>P. cubensis</i> (PcC1, PcC2, PcC3, PcC8, PcC11 and PcC12) were found to be infect all five hosts namely, cucumber, muskmelon, pumpkin, watermelon and summer squash and only two isolates (PcC9 and PcC19) were found to be non-pathogenic to all other hosts except cucumber suggesting that there might be the presence of different pathotypes in Punjab. This study confirms that isolates of <i>P. cubensis</i> are highly dynamic and the existence of considerable degree of diversity among them indicate that management is urgently required to reduce the losses incurred by smallholder farmers.</p>