<b>Objectives:</b> Vulvovaginal melanoma (VVM) is a rare but aggressive subtype of melanoma. VVM is considered to be less immunogenic and less responsive to immune-oncology therapy (IO) than cutaneous melanomas (CM). Vulvar and vaginal melanomas are typically grouped together due to the rare nature of the disease, though limited data suggest worse outcomes with vaginal melanomas. There is a paucity of data regarding molecular differences between these two melanoma subtypes. Our goal was to explore molecular profiles and survival between vaginal and vulvar melanomas. <b>Methods:</b> Samples were analyzed using next-generation sequencing (NGS) (TruSeq, 45 genes; NextSeq, 592 genes and NovaSeq, WES), IHC, and WTS (NovaSeq) (Caris Life Sciences, Phoenix, AZ). PD-L1 expression was tested by IHC using 28-8 (Agilent) and SP-142 (Spring Biosciences) (positive cut-off ≥1%). Microsatellite instability (MSI) was tested by fragment analysis (FA), immunohistochemistry (IHC), and NGS. Tumor mutational burden (TMB) was measured by totaling somatic mutations per tumor (TMB-high cut-off ≥10 mutations/MB). Immune cell fraction was calculated by QuantiSeq (Finotello 2019, Genome Medicine). Real-world overall survival (OS) was extracted from insurance claims data and calculated from the time of IO treatment to the last contact using Kaplan-Meier survival curves for molecularly defined cohorts. Statistical significance was determined using Chi-square and Wilcoxon rank-sum test and adjusted for multiple comparisons (q<0.05). <b>Results:</b> A total of 183 VVM were included in this analysis: 137 (74.9%) vulvar and 46 (25.1%) vaginal. Rates of gene mutations between these tumors were similar. Vaginal tumors trended toward increased frequency of <i>ATRX</i> (47.1 vs 23.2%) and <i>TP53</i> (29.0 vs 16.1%) mutations compared to vulvar tumors. Vulvar tumors trended toward higher rates of <i>SF3B1</i> mutations (33.8 vs 4%) as well as more <i>KIT</i> mutations (17.7 vs 8.8%) and amplification (16.7 vs 8.0%). PD-L1 expression was similar in vaginal and vulvar tumors (40 vs 32.7%). MMR deficiency and high TMB were absent in both groups. Vulvar melanoma trended towards increased immune checkpoint gene expression compared to vaginal melanoma, most notably a 2-fold increase in both <i>IFNG</i> and <i>IDO1</i> expression. No difference was seen in immune cell infiltrates. Treatment and survival data were available for 78 vulvar and 40 vaginal melanomas (OS: 34 vs 21 mo). In total, 15% of vulvar patients (<i>n</i>=12) and 18% of vaginal patients (<i>n</i>=7) received IO therapy. Median survival was similar between groups (vulvar 18 mo vs vaginal 19 mo, p=0.9). The survival of cutaneous melanoma (CM) patients receiving IO therapy (<i>n</i>=1047) was roughly twice as long (36.6 mo,p=0.038). <b>Conclusions:</b> This is one of the largest VVM cohorts to be studied to date. Overall, we found that these tumors were very similar in their molecular profiles and immunogenicity. Survival and response to IO therapy appeared similar between vulvar and vaginal melanomas. Our findings support continuing study of VVM as a single entity in light of the rarity of this disease.Fig. 1