The humid climate in the southeastern United States is conducive for bacterial and fungal diseases of peach. A survey of prematurely declining peach trees in South Carolina orchards revealed the presence of Cytospora spp isolates in many production fields. On potato dextrose agar the isolates revealed highly variable colony morphologies. To further investigate the isolates on the molecular level, we sequenced the ribosomal Internal Transcribed Spacer regions 1 and 2. Six genotypes were identified that formed distinct clusters within published Cytospora leucostoma Genbank entries. The isolates were sensitive to thiophanate-methyl and pyraclostrobin but resistant to boscalid. Preliminary results show two more isolates that most closely match Cystospora donestzica in Genbank, but more information is needed to confirm identity. Two peach cultivars were inoculated with one representative each of the eight genotypes and canker formation was monitored over four months. Koch’s postulates were confirmed for all isolates, but we found highly variable virulence among single tree replicates of the same cultivar. The variability could not be explained with deficiencies in soil nutrient content. Although there were a variety of different plant pathogenic nematodes in the soil, there were no statistical differences in the nematode count in soil of experimental tree replicates. By evaluating these isolates we gain a better understanding of the genetics and diversity of Cytospora species affecting peach trees in South Carolina.