Peach tree health, longevity, and limb strength can be affected by wood decay fungi, but the extent of the colonization and their identities have not been characterized in Georgia, United States of America. In an incubation experiment, dense white fungal mycelium grew on the cross-cut faces of asymptomatic limb sections sampled from peach scion cultivars, but no mycelium grew on those from rootstock cultivars. Among the scion cultivars, a Chi-square analysis indicated significant differences in the incidence of colonization. A dissection of six broken peach tree scaffold limbs showed symptoms of colonization along the entire length of the limbs. Only 41 % of primary branches had symptoms of wood decay, but no secondary branches appeared to have symptoms. Next generation sequencing (NGS) and metagenomic analysis of a combined DNA sample revealed the contigs were aligned to sequences of Trametes versicolor and/or Schizophyllum commune, suggesting these two white-rot fungi were present in the sample tested. Sanger sequencing confirmed two fragments of the same size but distinct nucleotide peak intensities were amplified from the sample used for NGS by two pairs of primers, providing supporting evidence that the two fungi were in the bulk sample but might differ in abundance or incidence. Subsequent multiplexing polymerase chain reaction diagnostics of ten scion samples confirmed the dominance of Trametes spp. (all ten samples), although Stereum spp. (five samples), Schizophyllum spp. (three samples) and Hericium spp, (one sample) were sometimes found coexisting with Trametes spp. Future research needs on this peach-fungus association are discussed.