Taproot decline (TRD), caused by an undescribed species of Xylaria, is an emerging root disease of soybean in Mississippi. Xylaria sp. isolates were collected from soybean roots and used to characterize TRD distribution as well as optimal growth temperature, pathogenicity, alternative host range, in vitro pathogenicity, and fungicide sensitivity. The 24 selected Xylaria sp. isolates from the 2016 collection had a mean optimal growth temperature of 26.7°C, and were pathogenic to soybean; however, differences in virulence occurred among isolates. Five selected Xylaria sp. isolates produced stroma on six hosts: corn, cotton, peanut, rice, sorghum, and wheat. Xylaria sp. colonized corn, cotton, and soybean seed in vitro; however, only reduced germination in soybean. Three selected Xylaria sp. isolates were exposed to fungicide-amended potato dextrose agar with concentrations up to 100 ppm of commercial products typically used in soybean production systems. Xylaria sp. isolates were insensitive to all active ingredients except thiophanate-methyl.