Previous studies have demonstrated the utility of fluorescence in situ hybridization (FISH) as an ancillary method in the diagnostic workup of histopathologically ambiguous melanocytic neoplasms. A combination of probes targeting 3 loci on chromosome 6 and 1 on 11q has been reported to distinguish unequivocal melanomas and nevi with a sensitivity and specificity of 87% and 96%, respectively. However, information on how FISH should be integrated into routine clinical testing is limited. We report our experience of FISH testing of 804 ambiguous melanocytic lesions performed as part of routine workup at University of California, San Francisco. The main category (47% of all cases) for which FISH testing was requested was Spitz tumors. Other categories included the distinction of possible melanoma from combined nevi (9%), acral or mucosal nevi (9%), Clark/dysplastic nevi (7%), and blue or deep penetrating nevi (6%) and to assess the possibility of nevoid melanoma (4%). Of the ambiguous tumors successfully tested, 88% received a more definitive benign or malignant final diagnosis. Of the 630 cases that tested negative by FISH, the final diagnosis was benign in 489 (78%) cases, ambiguous in 91 cases (14%), and malignant in 50 cases (8%). A positive FISH result was observed in 124 cases, with a final diagnosis of melanoma in 117 (94%). One (1%) FISH-positive case had an equivocal final diagnosis, and 6 (5%) were interpreted, despite the positive FISH result, as melanocytic nevi. We conclude that FISH testing can help reduce the number of equivocal diagnoses in ambiguous melanocytic neoplasms, in particular if FISH testing is positive, and discuss the challenges and limitations of FISH in clinical practice.