Anthracnose fruit rot was observed in some late-season peach cultivars in South Carolina in the 2012 and 2013 production seasons as well as increased anthracnose leaf spot of blueberry in a commercial operation of the same state in 2012. Single-spore isolates of Colletotrichum siamense were either sensitive or resistant to both thiophanate-methyl and azoxystrobin with the concentration of the fungicide at which fungal development is inhibited by 50% of ≥100 μg/ml. Resistant isolates revealed the E198A mutation in β-tubulin and the G143A mutation in cytochrome b. Nucleotide sequence analysis of the complete CYTB gene from genomic DNA of C. siamense isolates revealed an intronless genotype (CsI) and a genotype revealing two introns (CsII) at amino acid positions 131 and 164. Resistance to thiophanate-methyl or azoxystrobin was not found in isolates of C. fructicola collected from peach fruit. The CYTB gene of isolates of this species was of the CfII genotype or revealed a unique CfIIa genotype. Phylogenetic analysis of C. siamense isolates from different locations and different crops showed that the resistant isolates were genetically closer to each other than to sensitive isolates, suggesting that field resistance to thiophanate-methyl and azoxystrobin fungicides is derived from a common ancestor.