(1) Background: Cervical cancer, caused mainly by high-risk Human Papillomavirus (hrHPV), is a significant global health issue. While a Pap smear remains a reliable method for early detection, identifying new biomarkers to stratify the risk is crucial. For this purpose, extensive research has been conducted on detecting DNA methylation. (2) Methods: This cross-sectional study aimed to assess the expression levels of EIF4G3 and SF3B1 in precursor lesions and cervical tumor tissues through qRT-PCR and evaluate the methylation status of their promoters through bisulfite conversion. (3) Results: Both genes showed similar mRNA expression patterns, with the highest levels observed in squamous cell carcinoma (SCC) samples (p EIF4G3—p = 0.0016; SF3B1—p < 0.0001). (4) Conclusions: Regarding the methylation pattern for both genes, we observe a decreasing trend from NILM to SCC patients. Therefore, we concluded that the decrease in methylation at the promoter level for both genes could be an indicator of abnormal cytology.
