AIM: To analyze differentially expressed genes after multiple sevoflurane anesthesia in rhesus macaques was studied. METHODS: Rhesus macaques received three anesthetic exposures to sevoflurane on postnatal day (P) 7 and then on P21 and P35. RNA sequencing analysis was used in the studies. RESULTS: Transcriptomic analysis of differentially expressed genes (DEGs) revealed that up-regulated genes were mainly enriched in the regulation of positive regulation of gastrulation and odontogenesis, while down-regulated genes were primarily enriched in the regulation of transcriptional activator activity, RNA poly-merase II transcription regulatory region sequence-specific binding, and MAPK signaling pathway. CONCLUSION: Differentially expressed genes (DEGs) were analyzed in the prefrontal cortex of rhesus macaques after multiple sevoflurane exposures by RNA sequencing to screen essential genes for subsequent studies on neurotoxicity induced by sevoflurane, providing a solid basis for the study of the mechanism of general anesthesia.