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DNA methylation is an epigenetic modification of the genome, with methylation-based biomarkers showing promise for cancer detection. Quantitative Methylation-Specific PCR (qMSP) is widely used to assess DNA methylation; however, its application to liquid biopsy samples presents technical challenges. Heterogeneity in methylation patterns may impact qMSP accuracy by reducing reliability. This study evaluated the impact of methylation heterogeneity on qMSP performance using synthetic DNA fragments mimicking methylation variation. Additionally, targeted methylation sequencing was performed on prostate cancer tissue and urine samples to examine methylation heterogeneity. The presence of unmethylated CpG sites within the primer and probe regions reduced fluorescence levels and increased Cp values, especially at the 3'-end of primers. The methylation sequencing of genomic DNA from prostate cancer tissue and urine samples revealed the presence of varying methylation patterns, correlating with qMSP outcomes. Tissue samples mainly exhibited fully methylated and fully unmethylated fragments, while urine samples consisted of a higher proportion of partly methylated fragments. The findings suggest that qMSP may not be a reliable method for methylation detection in samples with proportionally high levels of heterogeneous methylated fragments in contrast to fully methylated fragments. This highlights the need for improved DNA methylation analysis in liquid biopsy samples for clinical applications in cancer detection.