Background <p>The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) gene promoter is pivotal in clinical decision-making for glioma patients. Pyrosequencing (PSQ) has been regarded as the gold standard for determining the MGMT promoter status. Nevertheless, PSQ is limited by its low throughput, high costs, and intricate protocols. In this study, we present a comparative analysis of the performance of PSQ and next-generation sequencing (NGS) in evaluating MGMT methylation in glioma patients.</p> Methods <p>Initially, we developed an amplicon-based NGS method for quantifying MGMT methylation. Subsequently, a comparative assessment was carried out to evaluate the MGMT promoter methylation levels in 50 formalin-fixed paraffin-embedded (FFPE) glioma samples using both PSQ and NGS. Finally, a consistency analysis was performed to compare the results obtained from PSQ and NGS.</p> Results <p>The results revealed a significant correlation between PSQ and NGS (R² = 0.88). Moreover, the consistency rate of the test results among the 50 samples was 94% (47/50), with one negative sample and two positive samples showing inconsistency. These three samples were verified using MethyLight technology, and the results were consistent with those obtained from NGS.</p> Conclusions <p>This study indicates that, although PSQ is the gold standard, the quantitative detection of MGMT methylation by NGS is more accurate than that by PSQ. NGS is characterized by high throughput and cost-effectiveness, while also yielding accurate and stable results. Therefore, NGS provides a viable alternative to the PSQ method for detecting MGMT methylation.</p>