Abstract: 【Objective】 This study aimed to identify autophagy-related genes （ATGs） in Neoscytalidium dimidiatum， the pathogen of pitaya canker， and analyze their expression changes under stresses of hydrogen peroxide （H₂O₂）， pyraclostrobin， and difenoconazole， providing theoretical basis for further research on their functions. 【Method】 ATGs were identified from the genome of Neoscytalidium dimidiatum， and bioinformatic analysis was performed. The transcriptome data analysis and real-time fluorescence quantitative PCR were utilized to detect expressions of ATGs of Neoscytalidium dimidiatum under stresses of H₂O₂， pyraclostrobin， and difenoconazole. Autophagic vacuoles were observed using microscopes， and the tolerance of Neoscytalidium dimidiatum to Congo red and carbon/nitrogen source deficiency was evalua-ted. 【Result】 Thirty ATGs were identified from the genome of Neoscytalidium dimidiatum， and phylogenetic analysis showed that most genes shared close relationships with homologous genes of Ascomycota fungi. Gene expression analysis revealed that oxidative stress （H₂O₂） exhibited weak inhibitory effects on regulating expressions of ATGs of Neoscytalidium dimidiatum， as it only induced five differentially expressed genes （one up-regulated and four down-regulated）. In contrast， both fungicides （pyraclostrobin and difenoconazole） mainly exhibited inhibitory effects， leading to 12 and 17 down-regulated genes respectively， and no up-regulated genes were detected in the two treatment groups. Microscopic observation showed that H₂O₂ had the weakest inhibitory effects on autophagic level of Neoscytalidium dimidiatum， while difenoconazole had the strongest. Colony assays demonstrated that the tolerance of Neoscytalidium dimidiatum to Congo red was reduced under pyraclostrobin stress； moreover， stress of the two fungicides reduced the tolerance of Neoscytalidium dimidiatum to carbon/nitrogen source deficiency. 【Conclusion】 Differentially expressed ATGs under oxidative and microbicide stresses play key roles in maintaining cellular homeostasis and responding to abiotic stresses for Neoscytalidium dimidiatum by regulating autophagy activity.