Pythium aphanidermatum is one of the most aggressive species in the genus and has a wide host range, but little is known about its population genetic structure. We tested 123 P. aphanidermatum isolates with six AFLP primer combinations and four SSR markers. The genetic diversity of P. aphanidermatum was 0.34 with AFLP and 0.55 with SSR markers. SSR genotypes totaled 3-8 for each locus, and a total of 14 SSR genotypes were found among all isolates. Three major genetic groups were identified with the combination of AFLP and SSR marker data. The genetic structure observed among P. aphanidermatum isolates was related to location and mefenoxam fungicide resistance instead of host. Four genotypes (PA1, PA2, PA5 and PA7) were found in the population from a commercial greenhouse, and the genetic diversity of a greenhouse population was similar to that found in the whole sample. The molecular tools for P. aphanidermatum isolates identified the possible gene flow within and among populations in Pennsylvania greenhouses.