Six simple sequence repeat (SSR)-enriched genome libraries from Pythium aphanidermatum, P. irregulare, and P. cryptoirregulare were constructed to develop SSR markers. One hundred six SSR primer pairs for P. aphanidermatum, 73 for P. cryptoirregulare, and 82 for P. irregulare were initially identified. After examining primers, the most polymorphic and reproducible SSR markers were selected for each Pythium species; 14 in P. aphanidermatum, 21 in P. irregulare, and 22 in P. cryptoirregulare. Analysis of isolates from each Pythium species using SSR markers showed the high degree of gene diversity and polymorphic information content (PIC) value in the three species. The average number of alleles was 3.5-5.3 in the three Pythium species. Seven SSR loci from P. cryptoirregulare and P. irregualre showed the distinct genetic separations of P. irregualre complex isolates. SSR markers identified for the three Pythium target species were highly transferable to other closely related Pythium species. Cross-amplification was found in all SSR markers between P. cryptoirregulare and P. irregulare. SSR loci were successfully amplified by direct PCR from mycelia of P. aphanidermatum, P. cryptoirregulare, and P. irregulare. These newly developed SSR markers can be used for population genetic studies and monitoring the movement of isolates in crop production systems or in nature.