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Anthracnose Fruit Rot (AFR) of strawberry, caused by <i>Colletotrichum acutatum</i>, is a major disease in Florida and frequent fungicide applications are needed for disease control. Quinone outside inhibitor (QoI) fungicides such as azoxystrobin and pyraclostrobin are commonly used to control AFR. From 1994 to 2014, 177 <i>C. acutatum</i> isolates were collected from multiple strawberry fields in Florida with or without a QoI spray history. Isolate sensitivity to both fungicides was tested based upon mycelial growth, germ tube elongation and fruit inoculation assays. Mean EC₅₀ values for isolates collected prior to 2013 based upon mycelial growth and germ tube elongation were 0.22 and 0.013 µg/ml for azoxystrobin and pyraclostrobin, respectively. Mycelial growth and germ tube elongation of 43 isolates collected from 2013 to 2014 were not inhibited at the highest concentrations of azoxystrobin (3 µg/ml) and pyraclostrobin (0.110 µg/ml). Azoxystrobin and pyraclostrobin sprayed preventively on detached strawberry fruit inoculated with <i>C. acutatum</i> failed to control resistant isolates. Isolates were characterized by sequencing of the <i>cytochrome b</i> gene and resistant isolates had the G143A amino acid substitution at the <i>cytb </i>locus. To our knowledge this is the first report of resistance to QoI fungicides in <i>C. acutatum</i>. Our findings indicate that development of AFR management practices is essential to limit selection and spread of QoI resistant populations of <i>C. acutatum</i>.