Downy mildew of spinach, caused by Peronospora effusa, is a major limitation for spinach production in the U.S. There are 17 reported races of P. effusa and many new races have emerged in recent years. Examination of the mechanism of evolution of new races that overcome deployed resistance is critical to continue using disease resistance as an effective management strategy. The objective of this study was to develop a mutagenesis protocol with EMS for P. effusa and select isolates with an altered virulence phenotype compared to the parental wildtype isolate. Sporangia of isolate UA0510C (race Pfs 13) were exposed to various concentrations of EMS and then used to inoculate a near isogenic line (NIL1) that is resistant to race 13. Untreated sporangia served as a control. None of the 1200 NIL1 plants evaluated showed infection when inoculated with untreated sporangia. However, three infected NIL1 plants were observed when the EMS treated sporangia were used for inoculation. The symptoms were observed on NIL1 plants 23-25 days after inoculation. The three putative mutants recovered had an altered virulence pattern compared to the parental isolate when inoculated onto 20 differential spinach lines. The mutants also were different from each other based on the disease phenotyping. Sequence variation between the wild-type isolate and the mutants with altered virulence phenotypes are being examined.