Abstract: Peanut root rot is a globally significant peanut disease, primarily caused by Fusarium oxysporum. Prothioconazole is a broad-spectrum and highly effective triazole fungicide capable of controlling various fungal diseases, including those caused by Fusarium species. To evaluate the resistance risk level of F. oxysporum to prothioconazole, the sensitivity of 104 F. oxysporum isolates collected from Henan, Shandong, Hebei, and Liaoning provinces, as well as Beijing City was determined using the mycelial growth rate method to establish a baseline sensitivity. Five and eleven prothioconazole-resistant mutants were obtained through fungicide adaptation (20 generations) and UV mutagenesis, respectively. The resistant mutant obtained through adaptation was further analyzed for resistance stability, biological fitness, and cross-resistance. The results showed that the sensitivity frequency distribution of 104 isolates followed a continuous unimodal curve with a normal distribution. The EC₅₀ values range from 0.30 to 6.84 μg/mL with a mean of (2.13 ± 1.19) μg/mL, it can serve as the baseline sensitivity of F. oxysporum to prothioconazole. The resistance in the mutants was stably inherited. Compared to their parental strains, the resistant mutants demonstrated a broader adaptation to temperature. While their sporulation capacity was comparable to that of the parental strains, conidial germination was significantly reduced, whereas pathogenicity was significantly enhanced. Cross-resistance assays indicated no significant cross-resistance between prothioconazole and carbendazim, pyraclostrobin, fludioxonil, difenoconazole, tebuconazole, or propiconazole. In conclusion, F. oxysporum poses a low-to-medium resistance risk to prothioconazole. Rotating prothioconazole with fungicides lacking cross-resistance is recommended in field applications to delay the development of resistance.