Soybean production in Pennsylvania has increased substantially over the past 20 years and is a highly valued field crop, together with corn. Soilborne pathogens such as Pythium spp. can contribute to soybean stand establishment issues, particularly under the conservation tillage practices that are common in the state. In this study, we collected soil samples from eight asymptomatic soybean-corn rotation fields across six counties in southeastern Pennsylvania between May and June 2012. Pythium spp. were isolated via baiting, and tested for aggressiveness on both soybean and corn using laboratory assays. In addition to our culture-based survey, we also assessed the use of cytochrome oxidase subunit 1 pyrosequencing as a culture-independent method for measuring Pythium spp. diversity from environmental samples. Diversity estimates were consistent between the culture-based and pyrosequencing datasets; however, important methodological biases inherent to culture-independent methods may have led to some differences. Our results show that several Pythium spp. previously characterized as soybean or corn pathogens are present in southeastern Pennsylvania, including Pythium irregulare, P. sylvaticum, and P. ultimum var. sporangiiferum, with isolates showing aggressive phenotypes in lab assays.