Background: Penile carcinoma is a rare malignancy. However, in developing countries the incidence rate is higher with approximately 6 cases per thousand men. mPC has a very poor prognosis and effective systemic treatments are an unmet need. Understanding the molecular alterations is essential for evaluating possible targets for more effective systemic therapies. In this context, we sought to evaluate the landscape of molecular alterations in mPC and their impact on clinical outcomes. Methods: We retrospectively identified and collected clinical data of patients (pts) with mPC who had received at least one prior systemic treatment from three Brazilian hospitals. Tumor samples were evaluated by next-generation sequencing (NGS) using a targeted panel (FoundationONE CDx), where 324 genes were analyzed in addition to tumor mutational burden (TMB) and microsatellite instability (MSI). PD-L1 expression and HPV 16/18 status were determined using immunohistochemistry (IHC). Clinical data were collected using standard templates. Continuous and categorical variables were summarized using descriptive statistics. Analyses of overall survival (OS) and progression-free survival (PFS) were performed using the Kaplan-Meier method. The significance level was set at 5%. All analyses were conducted using SAS version 9.4. Results: Twenty-three pts were identified, 18 pts had tumor samples analyzed by both NGS and IHC. The median age was 53.7 (range: 18-86) years; 16 pts (69.6%) received first-line systemic treatment, and 7 pts (30.4%) had progressive disease after neo- or adjuvant chemotherapy and did not receive any systemic treatment at the time of mPC. With a median follow-up of 14.3 months (95% CI: 9.7-20.1) the median OS and PFS from metastatic disease diagnosis were 12.7 (95% CI: 5.5-14.3) and 9.6 (95% CI: 5.5-14.3) months, respectively. Regarding the genomic alterations, 16 (88.9%) had some significant known somatic variants. TP53, TERT, CDKN2A, PIK3CA, NOTCH1, and CDKN2B loss were identified in 66.7%, 50%, 50%, 33.3%, 27.8%, and 22.2% of the pts, respectively. No MSI or TMB high (>10 mutations/MB) cases were identified. TMB low was identified in 83.3% of the pts. NOTCH1 mutation was the only one associated with worse OS (Yes: 5.5 vs No: 12.8 months, p=0.049), and PFS (Yes: 5.5 vs No: 11.7 months, p=0.032). Data on HPV status and PD-L1 expression status and its correlation with molecular alterations are currently being assessed. Conclusions: This translational study demonstrated that molecular alterations in mPC in developing countries are similar to those in patients from developed countries. Predictive biomarkers for immunotherapy, such as TMB high or MSI, were not identified in this study cohort. Specific gene mutations may identify patients with worse prognoses and open new avenues for therapeutic development.