Pythium oligandrumis an important potential biological control agent of plant pathogens. The objective of this study was to evaluate the genetic diversity and population structure of 45 isolates from various provinces in Iran. Genetic diversity was characterized based on inter-simple sequence repeat (ISSR) markers. Cluster analysis of ISSR data categorized the isolates into three main clusters. A high level of variability was revealed withinP. oligandrumisolates, however, no significant diversity among geographical regions was observed and isolates from different regions were grouped into the same cluster. Genetic diversity was moderate within populations (i.e. Bushehr, Fars and Kermanshah) with high average gene flow and low genetic distances between populations. The observed and effective numbers of alleles were higher in the Fars population than in other studied populations. This is the first report on the genetic diversity ofP. oligandrumusing ISSR markers. This study paves the way for the selection of isolates to be assessed for their antagonistic efficacy.