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Cercospora leaf spot (CLS) caused by Cercospora beticola is the most destructive foliar disease of sugar beet worldwide and is mainly controlled by timely fungicide applications. Recently, CLS control by pyraclostrobin exhibited significantly reduced efficiency in some fields of the Chifeng area in Inner Mongolia Autonomous Region of China. Purification of fungi by single spore isolation is essential for the study of fungal and fungicide resistance. However, the conventional single spore isolation and fungicide sensitivity assay for C. beticola are time‐consuming with a risk of contamination. We developed a new method for single spore isolation of C. beticola, which facilitated monitoring fungicide sensitivity of infected field samples. First, the composite spore suspension was prepared from typical lesions by vortex in the sterilized centrifuge tube. Then, the diluted spore suspension was spread on the streptomycin‐amended potato dextrose agar (PDA) plate or the PDA plate contained a threshold dosage of specific fungicide. The C. beticola colony derived from a single spore could be observed with naked eye and easily isolated by a sterilized scalpel knife after 36 h of incubation at 25°C. Meanwhile, the percentage of fungicide‐resistant or ‐tolerant isolates could be analysed based on the growth of different PDA media within 3 days. Based on this method, we identified that 22.88% of the C. beticola isolates were resistant to pyraclostrobin in the Chifeng area, indicating a high risk of emergence of C. beticola‐resistant strains to QoI fungicides in this area. Furthermore, the G143A or F129L mutation of the cytochrome b (Cyt‐b) gene was found in these resistant isolates. To our knowledge, this is the first report of C. beticola QoI‐resistant isolates with G143A or F129L mutation of CbCyt‐b gene in China. Our approach provides considerable potential for studying the biology, fitness and fungicide resistance of C. beticola. [ABSTRACT FROM AUTHOR]