Background: Molecularly targeted therapy is improving response rates and overall survival in subsets of melanoma patients. However, targeted therapy for “triple negative” patients (BRAF, NRAS and c-KIT wild type) is not yet defined. In addition, new evidence suggests that germline variants may have an impact in melanoma progression and response to therapy. In this study, we attempt to define the utility of a recently developed clinical assay that encompasses targeted sequencing of 50 genes with known impact on cancer progression. Methods: We used the AmpliSeq Cancer Panel HotSpot.V2 from Ion Torrent. The panel targets sequencing of Hotspot regions including 2,800 COSMIC mutations within 50 oncogenes and tumor suppressor genes. Tumor and blood germline DNAs were studied. All identified mutations were independently validated by Sanger sequencing. We then linked the molecular profile to extensive clinicopathological information including treatment and prospective clinical follow-up. Results: We examined 35 tumor samples from 33 melanoma patients (2 patients had 2 specimens). 14/35 (40%) had BRAF mutations, 12/35 (34%) had NRAS mutations (one patient had both BRAF and NRAS mutated) and 2 had c-KIT mutant tumor. Eight patients were triple negative and presented with mutations in KRAS (n=2), TP53 (n=5), ERBB4 (n=1), PI3K (n=3), RB1 (n=1), NOTCH (n=1), EZH2 (n=1), APC (n=5) and KDR (VEGFR2; n=2). We detected germline variants in TP53 (40%), KDR (34%), APC (29%), KIT (23%) and PIK3CA (14%) genes. Notably, KDR Q472H has been shown to affect VEGFR2 function. Patients with KDR Q742H had higher microvessel density, higher VEGF production and a shorter survival (15.1 months compared to 54.6 months). Conclusions: Sequencing using a validated clinical assay was informative of several targetable mutations in triple negative melanoma. Inhibitors targeting some of these mutations are already FDA approved for non-melanoma and others are currently tested in clinical trials. Our data also revealed a role of germline variant KDR Q472H in melanoma progression, that was not reported before, suggesting that further functional studies are warranted.