Abstract Aims To isolate and characterize fungi associated with diseased soybean seedlings in Midwestern soybean production fields and to determine the influence of environmental and edaphic factors on their incidence. Methods and Results Seedlings were collected from fields with seedling disease history in 2012 and 2013 for fungal isolation. Environmental and edaphic data associated with each field was collected. 3036 fungal isolates were obtained and assigned to 76 species. The most abundant genera recovered were Fusarium (73%) and Trichoderma (11.2%). Other genera included Mortierella, Clonostachys, Rhizoctonia, Alternaria, Mucor, Phoma, Macrophomina and Phomopsis. Most recovered species are known soybean pathogens. However, non-pathogenic organisms were also isolated. Crop history, soil density, water source, precipitation and temperature were the main factors influencing the abundance of fungal species. Conclusion Key fungal species associated with soybean seedling diseases occurring in several US production regions were characterized. This work also identified major environment and edaphic factors affecting the abundance and occurrence of these species. Significance and Impact of the Study The identification and characterization of the main pathogens associated with seedling diseases across major soybean-producing areas could help manage those pathogens, and devise more effective and sustainable practices to reduce the damage they cause.