Sentinel lymph node biopsies are conducted to stage patients with newly diagnosed melanomas that have histopathological attributes conferring defined levels of metastatic potential. Because benign nevic cells may also form 'deposits' in lymph nodes (nodal nevus), the pathological evaluation for metastatic melanoma within sentinel lymph nodes can be challenging. Twenty-eight sentinel lymph node biopsy cases containing either metastatic melanoma (N=18) or nodal nevi (N=10) were retrieved from the archives of the Brigham and Women's Hospital, Department of Pathology (2011-2014). In addition, two sentinel lymph node cases that were favored to represent metastatic disease but whose histopathological features were viewed as equivocal, with melanoma favored, were also included. Dual labeling for the melanocyte lineage marker, MART-1, and the epigenetic marker, 5-hydroxymethylcytosine, a functionally significant indicator that has been shown to distinguish benign nevi from melanoma, was performed on all cases using immunohistochemistry and/or direct immunofluorescence. All (18 of 18) metastatic melanoma cases showed complete loss of 5-hydroxymethylcytosine nuclear staining in MART-1-positive cells, and all (10 of 10) nodal nevus cases demonstrated 5-hydroxymethylcytosine nuclear staining in MART-1-positive cells. In addition, 5-hydroxymethylcytosine staining confirmed the favored diagnoses of metastatic melanoma in the two 'equivocal' cases. Thus, 5-hydroxymethylcytosine may be a useful adjunctive marker to distinguish between benign nodal nevi and metastatic melanoma during the evaluation of sentinel lymph node biopsies for metastatic melanoma.