The frogeye leaf spot pathogen of tobacco, Cercospora nicotianae, has three levels of sensitivity to azoxystrobin, the only synthetic, systemic fungicide currently labeled for tobacco fungal leaf spot management. Single nucleotide polymorphisms result in F129L and G143A mutations in the C. nicotianae cytochrome b gene, which confer moderate and high resistance, respectively. In some pathosystems, mutant pathogen individuals are less biologically fit than wild type individuals. To establish if there was a fitness cost in C. nicotianae with the mutant phenotypes, in vitro comparisons were made. Fitness was determined from conidial viability and mycelial growth rate in potato dextrose agar (PDA). In the mycelial growth assay, isolates were grown for 14 days and perpendicular diameters were measured. Conidial viability was measured by germination counts after 20-28 h incubation on PDA. Average values by sensitivity grouping were compared using one-way ANOVA. Growth rates and conidial viability for the wild type, moderate, and highly resistant groups averaged 105.67, 112.10, and 120.33 mm²/day, and 92.8, 94.8, and 94.1% germination, respectively. There were no statistically significant differences, suggesting no in vitro fitness cost associated with reduced sensitivities to azoxystrobin. This lack of fitness cost suggests populations composed of resistant C. nicotianae individuals may not rapidly return to wild type sensitivity levels in the absence of azoxystrobin use.