Herein, we wanted to explore the molecular landscape of mucosal melanoma from different sites and identify potential molecular targets for future therapy. Mucosal melanomas (N = 40) from different sites (conjunctiva, sinonasal cavity, rectum, and vagina) were investigated. Targeted next-generation sequencing along with Nanostring gene expression profiling was performed. Genetically, conjunctival melanoma was characterized by BRAF-V600E (30%) and NF1 mutations (17%). Mucosal melanomas at nonsun-exposed sites harbored alterations in NRAS, KIT, NF1, along with atypical BRAF mutations. When comparing the gene expression profile of conjunctival melanoma and nonsun-exposed mucosal melanoma, 41 genes were found to be significantly deregulated. Programmed death-ligand 1 (PD-L1) presented a significant sixfold upregulation in conjunctival melanoma compared to the other mucosal melanomas. While melanomas of the sinonasal cavity, vagina, and rectum are molecularly similar, conjunctival melanoma is characterized by a higher frequency of BRAF-V600E mutations and differential expression of several genes involved in the immune response.