Abstract: Round spot of Panax notoginseng (Burk) F. H. Chen is a destructive leaf disease, caused by Mycocentrospora acerina (R. Hartig) Deighton. It has serious impacts on the production and quality of the Chinese herbal medicine P. notoginseng. In order to evaluate the sensitivity of M. acerina to five sterol demethylation inhibitors (DMIs), the mycelium growth rate method was used to detect the sensitivity of forty-five strains to flusilazole, difenoconazole, diniconazole, propiconazole, and tebuconazole. The cross resistances were analyzed as well. The EC₅₀ values of flusilazole, difenoconazole, diniconazole, propiconazole, and tebuconazole ranged from 0.04 to 3.81 µg/mL, 0.18 to 6.72 µg/mL, 0.08 to 7.75 µg/mL, 0.44 to 11.38 µg/mL, and 0.46 to 29.85 µg/mL, respectively. The analysis of the frequency distribution range of the EC₅₀ values revealed that the mean EC₅₀ values of five fungicides to the most strains with unimodal continuous curve distribution were (0.99 ± 0.64), (1.77 ± 0.97), (2.37 ± 1.39), (2.61 ± 1.48) and (3.18 ± 1.58) µg/mL respectively. There was a sub-population with reduced sensitivity to five fungicides, and the strains showed significant cross-resistance to the five DMIs fungicides. The cross-resistance correlation index between flusinazole and difenoconazole was 0.989 which was the highest. The results provided the theoretical basis for the rational use of DMIs to control P. notoginseng round spot in Yunnan Province.