Southern stem canker disease caused by Diaporthe aspalathi has caused major production losses in soybean in the Southeast U.S. Disease symptoms include stem lesions, internal stem discoloration, interveinal chlorosis, and death in susceptible genotypes. Stem colonization defined by quantitative PCR assay and direct plating of stem segments on agar, internal discoloration, and lesion lengths were assessed for three D. aspalathi isolates differing in virulence on susceptible and resistant soybean genotypes. Disease symptoms were compared between an in vitro toxin bioassay and the toothpick inoculation assay. The most virulent isolate of D. aspalathi produced longest internal discoloration on the susceptible genotypes G810-2057. D. aspalathi colonized resistant and susceptible soybean genotypes and the three isolates differed in toxin production. The toxin bioassay conducted at two dilutions produced disease symptoms significantly different from the toothpick assay on most soybean genotypes indicating the two assays cannot act as substitutes in phenotype screening.