Abstract: Thiophanate-methyl, tebuconazole, prochloraz, and difenoconazole have been adopted for many years for the control of hickory canker (Botryosphaeria dothidea). In order to understand the resistance status and guide the prevention and control of hickory canker, the resistance frequencies of B. dothidea to the four fungicides were detected with the discrimination dose assay. EC₅₀ values of the four fungicides to some isolates were also investigated. The fitness differences in mycelial growth rate, number of pycnidia, and pathogenicity between thiophanate-methyl resistant isolates and sensitive ones were determined. The resistance mechanism of thiophanate-methyl resistant isolate was also analyzed. The results showed that most B. dothidea isolates have developed resistance to thiophanate-methyl with a resistance frequency of 84.37%. The average EC₅₀ value of thiophanate-methyl against the four sensitive B. dothidea isolates randomly chosen was 0.263 μg/mL and the relative resistance levels of five resistant isolates were between 49.9-84.8. All the tested isolates were sensitive to tebuconazole, prochloraz, and difenoconazole. The average EC₅₀ values of tebuconazole, prochloraz, and difenoconazole against six B. dothidea isolates chosen at random were 0.148, 0.023, and 0.091 μg/mL, respectively. In terms of fitness penalty, no significant difference in pathogenicity was detected between thiophanate-methyl resistant isolates and sensitive ones. However, the mycelial growth rates of the sensitive isolates were significantly faster than that of the resistant isolates, while the number of pycnidia of the sensitive isolates was significantly less than that of the resistant isolates. No mutations of amino acids related to benzimidazole resistance were detected in β-tubulin from isolates resistant to thiophanate-methyl.