The diagnosis of benign versus malignant melanocytic proliferations remains an important challenge. The current diagnostic algorithm typically involves an assessment of histopathologic and immunohistochemical parameters by light microscopy. Recently, fluorescence in situ hybridization (FISH) was reported as a useful ancillary diagnostic tool in melanocytic lesions, but the utility of FISH in melanocytic tumors that are difficult to diagnose with standard morphologic analysis remains controversial. To address this issue, we collected 34 ambiguous melanocytic tumors for FISH testing (NeoGenomics). Before FISH testing, cases were designated as "favor benign" (n=24) or "favor malignant" (n=10) by a consensus group (up to 7 dermatopathologists) on the basis of clinical, histopathologic, and immunophenotypic parameters. FISH was positive in 3/24 "favor benign" lesions and in 5/10 "favor malignant" lesions. The histopathologic, immunophenotypic, and FISH parameters informing our diagnostic impression were correlated with the final consensus diagnosis and clinical follow-up; in all cases, the initial diagnosis remained unchanged. In this series, the sensitivity of FISH for the histopathologic diagnosis of melanoma was 50%, the specificity was 87.5%, the positive predictive value was 62.5%, and the negative predictive value was 80.7%. Follow-up information was available for 25 patients (17 benign and 8 malignant diagnoses). Among benign lesions, the mean follow-up was 16.8 months (range, 8 to 25 mo); no metastases have been reported to date. Among the malignant lesions, the mean follow-up was 14.6 months (range, 7 to 23 mo); a single lymph node metastasis was identified in a 4-year-old girl with a histopathologic diagnosis of melanoma, which was negative on FISH. In our experience, in the setting of a lesion with predominantly benign findings, a negative FISH test-given its high specificity-is a reassuring finding that supports a benign diagnosis. In contrast, a positive FISH test should be carefully interrogated in the context of the complete histopathologic findings.