Isolates of Cercospora arachidicola were tested for sensitivity to the demethylation-inhibiting (DMI) fungicide tebuconazole using a microtiter plate assay and a new micro-colony assay. Sensitivities were determined based on EC50 values. Mutation of the target CYP51 gene, overexpression of CYP51, and ABC transporter activity as the possible mechanisms were investigated. Based on real-time PCR assays, there was no significant difference in CYP51 expression between tebuconazole-sensitive and resistant isolates, indicating that tebuconazole resistance was not associated with over-expression of CYP51. Except for one resistant isolate that became more sensitive, there was no apparent increase in tebuconazole sensitivity in the presence of ABC transporter inhibitors, suggesting that ABC transporter activity was not responsible for tebuconazole resistance in these isolates. However, alterations at codons 453 or 461 were revealed in CYP51 in four tebuconazole-resistant isolates. This is the first report of mutations in the CYP51 gene associated with DMI resistance in C. arachidicola.