Graminicolous downy mildews are economically important pathogens of grasses. These pathogens are most commonly found in tropical regions of Asia where crop losses can be devastating. Peronosclerospora philippinensis and P. sacchari cause destructive diseases of maize, while P. sorghi, the only Peronosclerospora species reported in the US, is a pathogen of sorghum. In this study we performed comparative genomics analyses on a selection of Peronosclerospora genomes for the development of a conventional multiplex PCR assay for the specific detection of the species of regulatory concern, P. philippinensis and P. sacchari. In addition, we designed internal control primers for Peronosclerospora at the genus-level and a plant internal control. The assay was optimized and evaluated against other graminicolous downy mildew pathogens. Sensitivity tests using environmental samples resulted in a limit of detection of 0.05 ng/?l to all the tested species. We observed no cross-reaction against closely related pathogens and other Peronosclerospora species. The conventional multiplex PCR assay we developed works effectively for the target species P. philippinensis/P. sacchari, P. sorghi and the internal Peronosclerospora genus and plant controls. This species-specific conventional multiplex PCR assay requires basic molecular laboratory equipment and techniques, making it ideal for the quick detection of many samples.