Abstract: In this work, the toxicity of difenoconazole to Colletotrichum fructicola at different development stages and its indoor control efficacy were determined. The sensitive baseline of C. fructicola to difenoconazole at major pear cultivation areas in South China (Hubei, Anhui, Jiangxi, Jiangsu, Zhejiang and Fujian Province) was established. The conidia germination and germ tube elongation of six representative strains treated by difenoconazole were determined by spore germination rate method. And its indoor efficacy was tested using detached pear leaves. The sensitivity of 120 strains of C. fructicola to difenoconazole was determined by mycelial growth rate method. The results indicated that difenoconazole had high inhibitory activities against mycelial growth and germ tube elongation. However, the inhibition against conidia germination of C. fructicola was weak, with the average EC₅₀ values of (0.33 ± 0.03) μg/mL, (2.61 ± 0.26) μg/mL and (29.87 ± 1.31) μg/mL, respectively. The protective and curative effects of 160 μg/mL difenoconazole to pear anthracnose were 92% and 79%, respectively. And those of 1 000 μg/mL carbendazim were 51% and 23%, respectively. 120 strains from major pear cultivation areas in South China were sensitive to difenoconazole, and the EC₅₀ values were in the range of 0.27-1.12 μg/mL. The frequency curves of EC₅₀ value was continuous and unimodal, and following the positive skewness distribution. Hence, the mean EC₅₀ value (0.59 ± 0.02) μg/mL could be recognized as baseline sensitivities of C. fructicola to difenoconazole. The study laid the foundation for the evaluation and monitoring of the field resistance of C. fructicola to the difenoconazole, and provided a theoretical basis for the application of this fungicide to control pear anthracnose in the pear cultivation area.