The use of molecular diagnostic methods such as fluorescence in situ hybridization (FISH) for challenging melanocytic neoplasms is becoming more widespread. In light of the diagnostic difficulty they pose, spitzoid melanocytic neoplasms constitute an area of greatest potential utilization. In this study we wished to evaluate the sensitivity of the currently used melanoma FISH probe assay in a group of unambiguous spitzoid melanomas. On the basis of comparative genomic hybridization data, copy number losses at chromosome 9 have long been recognized as a frequent event in melanoma. In this study we wished to evaluate the efficacy of 9p21 as a potential FISH target and then evaluate the added benefit of reflexing the standard melanoma FISH assay, with FISH targeting 9p21 and the centromere of chromosome 9 (Cep9). Cep9 was included as a control to exclude inadequate hybridization or truncation as a source of homozygous deletions. We first studied a training set of 85 melanomas and 58 nevi with FISH targeting 9p21 and Cep9. As per previous methodology, 30 cells were enumerated. In the training set, the nevi had a mean number of cells with homozygous 9p21 loss of 0.97, with a standard deviation of 1.26. The melanomas had a mean of 7.1 and a standard deviation of 6.76. This difference was significant (P=2×10−12). On the basis of the training set, we identified a cutoff of 10 homozygous deletions to distinguish between melanoma and nevi. In a subsequent validation set consisting of 76 melanomas and 88 nevi, we found this cutoff to have a sensitivity of 33% and a specificity of 100%. Finally, in our group of 43 unequivocal spitzoid melanomas, standard FISH against chromosomes 6 and 11 was 70% sensitive. Homozygous 9p21 loss was present in 11 of 27 (41%) cases tested. By combining the standard melanoma FISH assay with the 9p21 FISH assay, a combined sensitivity of 85% was found. Among these 27 cases tested with 9p21, there were 7 cases that were negative by the standard melanoma FISH assay but that were positive by 9p21, suggesting that the 9p21 assay may be highly complementary to the standard melanoma FISH assay. Hence, in this study, we validated the efficacy of 9p21/Cep9 as a diagnostic FISH assay in melanoma, and demonstrated its complementary effect to the standard FISH assay. 9p21 may be particularly helpful in lesions with spitzoid morphology.