Abstract: Camellia oleifera is an important woody oil-bearing tree species in China of significant economic value. To expand the approaches for variety improvement and to accelerate the development of germplasm resistant to anthracnose, this study employed chemical mutagenesis breeding using seeds of the 'Xianglin 210' cultivar. The seeds were treated with a gradient of ethyl methanesulfonate (EMS) concentrations (2%, 3%, and 4%) for durations of 3, 6, and 9 hours, respectively. Following two years of field observation, we assessed emergence rate, seedling growth indices, and key physiological and biochemical parameters, including the activities of superoxide dismutase (SOD) and peroxidase (POD), as well as the contents of malondialdehyde (MDA) and total flavonoids (TF). Putative resistant mutants were further identified through in vitro inoculation with Colletotrichum fructicola. Additionally, the expression levels of disease resistance-related genes (CoSOD1, CoPOD, CoIDD4, CoWRKY78) were analyzed among the selected mutants.Our results showed that the treatment with 2% EMS for 3 hours did not significantly inhibit emergence rate or seedling growth. In contrast, other treatment combinations led to significantly reduced emergence rates and impaired plant growth, with the effects intensifying with higher concentrations and longer exposure times. The 4% EMS treatment for 3 hours was identified as closest to the half-lethal dose (LD₅₀). This treatment also significantly altered the activities of SOD and POD, as well as the contents of MDA and TF. From a population of 71 EMS-mutagenized plants, we successfully screened and identified one mutant (C20) with strong anthracnose resistance and two mutants (C59 and C92) that were highly susceptible. Variations in SOD and POD activities were key determinants of the disease-resistant phenotype, and the analyzed disease resistance-related genes exhibited significant expression differences among the mutants.In conclusion, this study demonstrates the successful creation of anthracnose-resistant C. oleifera germplasm through EMS mutagenesis. The identification of an optimal treatment condition approximating the LD₅₀ provides a valuable reference for future chemical mutagenesis breeding programs in this species and contributes to enriching its germplasm resources.