Malignant melanoma of the vulva and vagina is relatively uncommon and accounts for <5% of all melanomas in women. The aim of our study was to establish the biological properties and evaluate potential therapeutic targets in these tumors. We collected a series of 65 cases from three centers and re-evaluated the tumor tissue for predominant growth pattern (superficial spreading, nodular, and mucosal lentiginous) and tumor thickness. KIT (CD117) expression was detected immunohistochemically. In addition, tumors were screened for BRAF, NRAS, and KIT mutations by PCR and DNA sequencing as well as for KIT amplifications by fluorescence in situ hybridization. None of the cases contained BRAF mutations. NRAS mutations and KIT amplifications were detected in similar frequency (∼12%) in tumors of the vulva and vagina. In contrast, KIT mutations were present in 18% of primary melanomas of the vulva, but in none of the tumors arising in the vagina. Moderate or strong KIT protein expression was detected in 30 cases, including all tumors with KIT mutations and 6 of the 7 with KIT amplifications. In conclusion, BRAF mutations are virtually absent in melanomas originating from the vulva or vagina, whereas NRAS mutations and KIT amplifications occur in both locations. KIT mutations appear to be specific for melanomas of the vulva, suggesting that in spite of the anatomic proximity, the development of vulvar and vaginal melanomas involves different molecular alterations which may be targeted by novel treatment approaches.