<p>Aberrant DNA methylation is a hallmark of nasopharyngeal carcinoma (NPC) pathogenesis. The aberrant DNA methylation patterns in NPC, particularly in its cancer stem cells (CSCs), and their underlying significance require further elucidation. We integratively performed DNA methylome and transcriptome combined with single-nucleus RNA sequencing to investigate DNA methylation and gene expression patterns of NPC and CSCs. Unlike Epstein-Barr virus (EBV)-negative cells, NPC and CSCs harboring EBV displayed global DNA hypermethylation and they were more oncogenic and immunosuppressive. By correlating DNA methylation and gene expression profiles, we disclosed potential relationships between aberrant DNA methylation, tumorigenesis, metastasis, immunotherapy response, and radiotherapy resistance of NPC. After validating with datasets from GEO and TCGA, we identified aberrant DNA methylation-associated biomarkers including 9 NPC-specific diagnostic markers that had significantly higher DNA methylation levels in NPC than in normal tissues and 8 types of cancers, and 12 potential prognostic markers that were highly correlated to cell cycle dysregulation. Notably, 2 of these potential biomarkers highly expressed in CSCs were validated at the single-cell level. Our study not only identified new potential diagnostic and prognostic biomarkers but also provided new insight into aberrant DNA methylation-associated pathogenesis of NPC, which is beneficial for the development of precision diagnosis and treatment schemes.</p>