Anthracnose disease caused by Colletotrichum spp. is major concern in strawberry production nationwide. A total of 200 Colletotrichum spp. isolates were obtained from strawberry plants in the Mid-Atlantic region of U.S. Analysis of partial g3pdh, tub2, and/or ITS sequences showed that four Colletotrichum species, C. nymphaeae, C. fioriniae, C. siamense, and C. lineola were associated with strawberry anthracnose, with C. nymphaeae being predominant. All isolates were tested for resistance to quinone outside inhibitor (QoI) fungicide azoxystrobin. C. siamense isolates were additionally tested for resistance to benzimidazole carbamate (MBC) fungicide thiophanate-methyl. Overall frequency of resistance to QoIs and MBCs was 48 % and 67 %, respectively. G143A or F129L mutation in the cytochrome b gene (cyt b) and E198A mutation in the β-tubulin (tub2) gene were found to be linked with respective resistance phenotypes. Moreover, microsatellite primers were selected for determining genetic diversity within C. nymphaeae populations, followed by fragment sizing and preliminary down-stream analysis.