A 4-color fluorescence in situ hybridization (FISH) assay, using probes to chromosomes 11q, 6p, 6q, and 6 cent, has recently been proposed as an ancillary tool for the diagnosis of melanoma. The authors report herein their experience with this assay. To determine the sensitivity and specificity of the assay for histopathologically unequivocal cases, they analyzed 50 melanocytic nevi, 50 primary melanomas, and 15 metastatic melanomas. Of 50 melanocytic nevi, 47 were FISH negative on initial readout (test sensitivity, 94%); 49 were FISH negative after correction for tetraploidy (test specificity, 98%). Of 50 primary melanomas, 41 were FISH positive (test sensitivity, 82%). Of 15 metastatic lesions, 13 were FISH positive (test sensitivity, 85%). Of the 9 FISH-negative melanomas, 6 metastasized. The tumors of the 5 patients who had survived thick primary melanoma for more than 5 years without recurrence were all FISH positive. Half of the patients whose primary melanoma was tested by FISH had undergone sentinel lymph node (SLN) biopsy. When the authors compared the FISH results of those 25 melanomas with the SLN status, no statistically significant correlation was found. These findings document limitations of the current FISH assay. A rare nevus may be FISH positive. Some primary metastasizing melanomas are FISH negative. Even metastatic melanomas can be FISH negative. Awareness of the limitations in test sensitivity and specificity of the FISH assay is important to avoid an erroneous diagnosis by overreliance on cytogenetic findings. Correlation with clinical and histopathological findings is paramount for accurate diagnosis.