The histologic differential diagnosis between a second primary cutaneous melanoma and cutaneous melanoma metastasis in a patient with a previous history of melanoma can be very difficult. This case report describes the first application of CDKN2A mutation analysis for discriminating a cutaneous melanoma metastasis from a new primary melanoma. In 2005, we received a skin excision of the right arm of a 38-year-old female patient for second opinion. Histologically, we considered the lesion to be a melanoma. The patient had a history of superficial spreading melanoma in the right subclavicular region, with a Breslow thickness of 1.1 mm, in 1998. The morphology showed resemblance to the present melanoma on the right arm, but the differential diagnosis between metastasis or second primary melanoma could not be made with certainty based on histology alone. We decided to perform TP53 and CDKN2A mutation analysis on both tumors. Molecular analysis revealed that both the melanoma of 1998 and of 2005 contained an identical CDKN2A mutation (a deletion in exon 1alpha, c.95_112del (p.Leu32_Leu37del)), which was absent in normal control tissue of the patient, thereby excluding a germline mutation. TP53 mutations were absent in both tumors and in normal skin. Based on these molecular findings the present melanoma on the right arm was diagnosed as a metastasis. Seven months later the patient died of widespread metastatic disease confirming the metastatic nature of the lesion. This case illustrates that molecular analysis can contribute to the sometimes-difficult differentiation between a second primary melanoma and a melanoma metastasis.