Abstract: The sensitivity of Colletotrichum gloeosporioides from strawberry to pyraclostrobin, a Qo inhibitor (QoI) fungicide, was assessed through the mycelial growth rate method and the relationship between alternative respiration and sensitivity to pyraclostrobin during mycelial growth in C.gloeosporioides was analyzed. The results showed that the medium effective concentrations (EC50Q) of 65 C.gloeosporioides isolates collected from strawberry fields in Lin'an, Jiandei and Zhuji of Zhejiang province ranged from 0.09 to 24.26 μg/mL, with an average value of 6.65 μg/mL in the absence of SHAM, a specific inhibitor of alternative oxidase. In the presence of 50 μg/mL SHAM, the EC50 (Q+S) ranged from 0.06 to 48.98 μg/mL, with an average value of 10.58 μg/mL. The factor value F=EC50Q / EC50 (Q+S) for the impact of alternative respiration on the sensitivity of isolates to pyraclostrobin ranged from 0.02 to 11.17, with an average value of 0.96; and the F value for 78.46% isolates was less than 1. The transcript levels of alternative oxidase encoding gene, AOX, in mycelia of strawberry and grape C.gloeosporioides isolates with different sensitivity to pyraclostrobin were studied through real-time quantitative PCR. The results indicated that pyraclostrobin significantly increased the AOX transcript levels after treatment for 6 to 12 h in both strawberry and grape isolates. However, no significant correlation between the AOX transcript levels and the sensitivity to pyraclostrobin was observed. The results of present study suggested that the alternative respiration pathway in different fungal pathogens is complicated.