Few fungicides are effective against anthracnose, caused by Colletotrichum spp., and emerging resistance makes the search for chemical alternatives more relevant. Isolates of the Colletotrichum acutatum species complex were collected from South Carolina and Georgia peach orchards and phylogenetic analysis of the combined internal transcribed spacer region, glyceraldehyde-3-phosphate dehydrogenase, and β-tubulin gene sequences separated the isolates into C. nymphaeae and C. fioriniae. The sensitivity of these and three other previously reported Colletotrichum spp. from peach, including C. fructicola, C. siamense, and C. truncatum, to demethylation inhibitor (DMI) fungicides difenoconazole, propiconazole, tebuconazole, metconazole, flutriafol, and fenbuconazole was determined based upon mycelial growth inhibition. C. truncatum was resistant to tebuconazole, metconazole, flutriafol, and fenbuconazole and C. nymphaeae was resistant to flutriafol and fenbuconazole based on 50% effective concentration (EC50) values >100 μg/ml. C. fructicola and C. siamense were sensitive to all DMI fungicides (EC50 values of 0.2 to 13.1 μg/ml). C. fioriniae subgroup 2 isolates were less sensitive to DMI fungicides (EC50 values of 0.5 to 16.2 μg/ml) compared with C. fioriniae subgroup 1 (EC50 values of 0.03 to 2.1 μg/ml). Difenoconazole and propiconazole provided the best control efficacy in vitro to all five species, with EC50 values of 0.2 to 2.7 μg/ml. Tebuconazole and metconazole were effective against all Colletotrichum spp., except for C. truncatum. The strong in vitro activity of some DMI fungicides against Colletotrichum spp. may be exploited for improved anthracnose disease management of peach.