Melanoma accounts for most skin cancer-related deaths and has an increasing incidence. Accurate diagnosis and distinction from atypical nevi can be at times difficult using light microscopy alone. Fluorescence in situ hybridization (FISH) and melanoma gene expression score (myPath, Myriad Genetics) have emerged as ancillary tools to further aid in this differential diagnosis. Our aim in this study was to correlate FISH results, gene expression score, consensus histopathologic impression and clinical outcome on a series of 117 challenging melanocytic lesions collected from three separate institutions. The lesions were separated into two groups: 39 histopathologically unequivocal lesions (15 malignant, 24 benign) and 78 challenging lesions interpreted by expert consensus (27 favor malignant, 30 favor benign, and 21 ambiguous). Melanoma-FISH was performed using probes for 6p25, 11q13, 8q24, and 9p21/CEP9 and scored according to established criteria. Analysis by myPath gene expression score was performed and interpreted by the manufacturer as 'benign', 'indeterminate,' or 'malignant'. In the unequivocal group, melanoma-FISH and myPath score showed 97 and 83% agreement with the histopathologic diagnosis, respectively, with 93 and 62% sensitivity, 100 and 95% specificity, and 80% inter-test agreement. In the challenging group, FISH and the myPath score showed 70 and 64% agreement with the histopathologic interpretation, respectively, with 70% inter-test agreement and similar sensitivities and specificities. The inter-test agreement was 73% overall, excluding indeterminate results. Discordant test results occurred in 27/117 cases from both unequivocal and challenging groups. Melanoma-FISH and gene expression score are valuable ancillary tools, though both have limitations and return discordant results in a subset of cases. Follow-up studies with more extensive clinical outcome data are warranted to establish the accuracy of these tests for the classification of melanocytic lesions.