BACKGROUND Clinical and dermatoscopic guidelines are used to differentiate between benign longitudinal melanonychia (LM) and subungual melanoma; however, the frequency of malignancy among patients undergoing a biopsy for LM is not well defined. OBJECTIVE To describe the histologic diagnoses and malignancy among patients undergoing a biopsy for clinical LM. METHODS Retrospective cohort study of consecutive patients who underwent a nail biopsy for LM at a single cancer center between 2000 and 2014. Clinical features, biopsy techniques, and histopathologic results were reviewed. RESULTS Forty-two patients with 43 biopsied lesions were included. Three of the 43 biopsies revealed melanoma (mean depth 2.1 mm). The mean age among patients with malignant lesions was 60 years compared with 58.1 years for benign lesions. The mean width of all biopsied lesions was 4.2 mm, with a mean of 10.7 mm for malignant and 3.4 mm for benign. The first digit was the most commonly involved nail in both malignant and benign lesions. CONCLUSIONS Most nail biopsies performed for LM revealed benign pathology; however, melanoma was diagnosed in a small subgroup. Although clinical and dermatoscopic guidelines help guide biopsies, they should not replace clinical judgment as malignant lesions can deviate from these guidelines.