Background: Oral Squamous Cell Carcinoma (OSCC) is characterized by an increasing incidence and a 60% 5-year survival. The most important prognostic factor for OSCC is the presence of lymph node (LN) metastases but the detection of LN metastases in the clinic by palpation and imaging is inaccurate resulting in under and overtreatment of patients. To improve LN diagnosis new detection methods are needed. DNA methylation studies can be used to identify novel biomarkers, as epigenetics have been established as an important regulator of metastatic potential. The aim of this study is to identify new DNA methylation markers that predict LN metastasis in OSCC. Materials and Methods: Genome-wide methylation patterns of metastatic (n = 6) and non-metastatic OSCC (n = 6) were assessed using MethylCap- Seq. Subsequently, analysis was performed on the most differentially methylated loci to identify pathways and genomic loci associated with LN metastasis. Additionally, the methylation data were combined with expression data of 222 OSCC patients acquired by an expression microarray with a 696 gene panel associated with N-status in OSCC. Finally, we validated these findings on the OSCC samples (n = 174) in The Cancer Genome Atlas (TCGA). Results: We found that genes on chromosome 7 are the most hypermethylated in metastatic OSCC in comparison to non-metastatic OSCC, more specifically a four million bp loci around the EGFR gene. In total 26 genes were found to be differentially methylated by MethylCap- Seq as well as differentially expressed by microarray. In the OSCC TCGA validation cohort, all 26 genes are significantly differentially expressed between metastatic and non-metastatic OSCC. For these 26 differentially expressed genes 88 probes were found in the TCGA Infinium 450k methylation data that are both significantly differentially methylated between the two groups and correlated with mRNA levels. Finally, six of these probes overlapped with the genomic regions annotated by MethylCap-Seq, representing five genes. Conclusion: We identified chromosomal loci, pathways and gene promoters associated with LN metastasis in OSCC patients. For 10 genes the differential methylation, expression and correlation between methylation and expression was validated on an independent OSCC cohort from the TCGA database. Increased understanding of the metastatic OSCC methylome will contribute to the identification of DNA methylation markers, pathways and potential therapeutic targets in OSCC primary tumors that will improve chances of providing the proper lymph node treatment and may result in improvement of survival and quality of life.