Abstract Background: The High-Resolution Melting (HRM) technology has recently been introduced as a rapid and robust analysis tool for the detection of DNA methylation. The methylation status of multiple tumor suppressor genes may serve as biomarkers for early cancer diagnostics, for prediction of prognosis and for prediction of response to treatment. Therefore, it is important that methodologies for detection of DNA methylation continue to evolve. Sensitive Melting Analysis after Real Time - Methylation Specific PCR (SMART-MSP) and Methylation Sensitive - High Resolution Melting (MS-HRM) are two methods for single locus DNA methylation detection based on HRM. Methods: Here, we have assessed the quality of DNA extracted from 30-years-old Formalin Fixed Paraffin Embedded (FFPE) tissue for DNA methylation analysis using SMART-MSP and MS-HRM. The quality assessment was performed on DNA extracted from 54 Non-Small Cell Lung Cancer (NSCLC) samples derived from FFPE tissue, collected over 30 years and grouped into five-years-intervals. For each sample, the methylation level of the CDKN2A (p16) and RARB promoters were estimated using SMART-MSP and MS-HRM assays designed to assess the methylation status of the same CpG positions. This allowed for a direct comparison of the methylation level estimated by the two methods for each sample. Results: The methylation level of the CDKN2A promoter was successfully determined by SMART-MSP and MS-HRM in all of the 54 samples. The same methylation estimates were obtained by the two methods in 46 of samples. The methylation level of the RARB promoter was successfully determined by SMART-MSP in all of samples. When using MS-HRM to assess RARB methylation five samples failed to amplify and 15 samples showed a melting profile characteristic for heterogeneous methylation. Out of the remainder 34 samples, for which the methylation level could be estimated, 27 gave the same result as observed when using SMART-MSP. Conclusion: MS-HRM and SMART-MSP can be successfully used for single locus methylation studies of DNA derived from up to 30-years-old FFPE tissue samples. Furthermore, it can be expected that MS-HRM and SMART-MSP will provide similar methylation estimates when assays are designed to analyze the same CpG positions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4918.