Damping off of soybean and corn, caused by Pythium spp., is favored by cool temperatures and wet soil conditions and is primarily managed using fungicide seed treatments. The goal of this research was to determine the effect of temperature on aggressiveness and fungicide sensitivity of Pythium spp. recovered from soybean and corn in Iowa. A total of 21 isolates of four of the most prevalent Pythium spp. in Iowa were screened. Seed and seedling assays were used to quantify the aggressiveness of P. lutarium, P. oopapillum, P. sylvaticum, and P. torulosum on soybean and corn at 13, 18, and 23°C. Isolates recovered from soybean or corn were equally pathogenic on both hosts. P. torulosum was more aggressive at 13°C compared with 18 and 23°C. Conversely, P. sylvaticum was more aggressive at 18 and 23°C than at 13°C. A plate assay was used to assess fungicide sensitivity to seven fungicides that are commonly used as seed treatments, and EC50 values at each of the three temperatures were determined and compared. EC50 values for P. torulosum were higher for all fungicides tested at 13°C, compared with 18 or 23°C, whereas EC50 values for P. sylvaticum were higher for all fungicides at 18 and 23°C compared with 13°C. These data contribute to our understanding of the effect of soil temperature on the risk of soybean and corn damping off, which may aid in the development of more effective management practices.