Soybean root rot is a devastating disease throughout the U.S. and Canada that reduces yield due to decreased absorption of plant nutrients. This disease is caused by Fusarium species and other common soilborne fungi. Recently, Fusarium proliferatum (FPR) has gained attention as a contributor to seedling disease and root rot in soybean. However, little is known about the relative aggressiveness of FPR as a root rot pathogen, especially pertaining to the environmental and ecological parameters influencing the ability of this species to cause disease. Fifty-two seedborne and seedling-associated FPR isolates from Kansas were collected and characterized using in vitro techniques to determine the optimum pH, temperature, and water potential tolerance ranges for fungal growth and pathogenicity. FPR isolates were characterized morphologically and by PCR to confirm species assignment. Thirteen of the seedling isolates were assayed using a rolled-towel pathogenicity test at 25-27°C and a pH of ~6.5. Seedling isolates reduced germination by 18% and reduced root length by 52% compared to mock-inoculated controls. Similarly, seedborne FPR isolates were shown to be pathogenic in both rolled-towel and greenhouse pot assays. In addition, niche width experiments that measure the variety of carbon sources used are in progress. The results provide foundational information that further studies can build upon to develop better field management practices against root rot in soybeans.