The diagnosis of certain melanocytic proliferations remains one of the most challenging areas in pathology. In recent times, fluorescence in situ hybridization (FISH) has emerged as a promising diagnostic aid to conventional microscopy. We previously showed that a 4-probe FISH assay targeting 6p25 (RREB1), 6q23 (MYB), Cep6 (centromere 6), and 11q13 (CCND1) could discriminate between histologically unequivocal melanomas and benign nevi with a sensitivity of 86.7% and specificity of 95.4%. However, the sensitivity of the assay is approximately 70% in melanomas with spitzoid morphology. Furthermore, differentiating true gains from tetraploidy may cause difficulties in interpretation by inexperienced examiners. Here we refine the current probe set to better target spitzoid melanomas and more easily distinguish cells with imbalanced copy number aberrations from tetraploid cells. Using FISH data from 3 training sets of 322 tumors, including 152 melanomas and 170 nevi, we identified 9p21, 6p25, 11q13, and 8q24 as a probe set with improved discriminatory power in differentiating melanomas from nevi. In a validation set of 51 melanomas and 51 nevi this probe set had a sensitivity of 94% and specificity of 98%, compared with the original probe set that had a sensitivity of 75% and specificity of 96% in the same validation cohort. We propose that by incorporating 9p21 into the 4-probe FISH assay, with a new diagnostic algorithm, this new probe set would have improved discriminatory power in melanocytic neoplasms and improved sensitivity for detecting spitzoid melanomas, as demonstrated by our previous studies.