Pestalotiopsis microspora is the cause of the nut black spot of Chinese hickory (Carya cathayensis), which seriously threatens nut production in China. In this work, the baseline sensitivity of P. microspora to pyraclostrobin, a QoI fungicide, was studied and highly resistant isolates (HRI) to pyraclostrobin were characterized biologically and pathologically. Pyraclostrobin showed a high activity against P. microspora in vitro, with effective median concentrations (EC₅₀) of 0.92 mg l⁻¹ and 0.08 mg l⁻¹ for mycelial growth and conidium germination inhibition, respectively. The EC₅₀ determined for a total of 165 isolates of P. microspora collected from three geographical regions of China between 2007 and 2009 varied from 0.01 to 0.25 mg l⁻¹ (mean of 0.13 ± 0.04 mg l⁻¹) following a unimodal distribution. Five HRI to pyraclostrobin, obtained after expose the mycelium to UV light, were cross-resistant to azoxystrobin, another QOI fungicide. However, no cross-resistance was obtained against boscalid, a fungicide having a different mode of action. Interestingly, the HRI were less virulent than their sensitive wild parents, but no difference between HRI and their sensitive wild parents were obtained in mycelia growth, conidial production and conidial germination. These results indicated that pyraclostrobin might be a good fungicide alternative to control the nut black spot of Chinese hickory.