The sensitivity of conidial germination and mycelial growth of Didymella bryoniae to azoxystrobin was compared to determine if mycelial growth assays can be used as an alternative to conidial germination fungicide sensitivity assays. Based on the dose response of mycelial growth, the azoxystrobin concentration of 1.0 g/ml was selected as a discriminatory concentration in mycelial growth assays. An allele-specific PCR assay was also developed to rapidly detect azoxystrobin resistance in D. bryoniae. Resistance to azoxystrobin was determined to be stable. Additionally, the relative fitness of azoxystrobin-, boscalid-, penthiopyrad-, and thiophanate-methyl-sensitive and resistant isolates was similar. However, thiophanate-methyl-resistant isolates grew significantly less than thiophanate-methyl-sensitive isolates at certain temperatures suggesting a fitness cost may be sustained. The results of this study will be used to rapidly and efficiently monitor fungicide resistance and to improve current fungicide resistance management recommendations.