Cercospora beticola is the most devastating foliar pathogen of sugar beet. This pathogen is primarily managed by the application of fungicides, including demethylation inhibitors (DMIs). Given the broad use of DMIs in managing Cercospora leaf spot, resistance has developed. Recently, five haplotypes of CbCyp51 have been correlated with DMI resistance. To improve our understanding of these haplotypes, this study has evaluated CbCyp51 expression across haplotypes with and without DMI exposure using RT-qPCR. Significant differences were found between haplotypes in control and difenoconazole groups, but the broader implications were unclear. This study also produced mutant C. beticola strains with replaced CbCyp51 haplotypes. DMI sensitivity was assessed, revealing dramatic changes in difenoconazole resistance. Most mutants exhibited elevated tetraconazole resistance, which was largely shown to be associated with the transformation process rather than haplotype exchange. Further studies involving the different haplotypes and mutants could improve our understanding of these DMI resistance associated mutations.