Background: Penile cancer is a rare disease associated with HPV infection and is known to harbor recurrent somatic genomic alterations in the ERBB (HER)-family, CDKN2A, TP53, NOTCH1 and PIK3CA. ctDNA assay allows the noninvasive genomic profiling of malignancies and may assist with understanding molecular evolution of resistance. To our knowledge, the genomic alterations observed in ctDNA for penile cancer have not been described before. We report ctDNA profiling of patients with advanced penile cancer. Methods: Sixteen pts with metastatic penile cancer from multiple institutions in the United States that underwent ctDNA analysis using the Guardant360 platform were eligible. Three patients had at least one serial ctDNA sample. De-identified demographic data were collected. Guardant 360 is CLIA-certified ctDNA panel that assesses single nucleotide variants and copy number alterations in 68 to 73 genes for potentially actionable genomic alterations. Variants seen at least 3 times in the Catalogue of Somatic Mutations in Cancer (COSMIC) database or reported in OncoKB were considered pathogenic. Results: The median age was 64 years (range 40-77). 4 pts (25%) were documented to be post platinum-based chemotherapy. The median number of ctDNA alterations detected per sample was 2 (range 0-6). Overall, genomic alterations were detected in 15/16 patients (94%) across 21 genes (table 1). Alterations were most frequently detected in TP53 (9/16, 56%), CDKN2A (5/16, 31%), TERT promoter (5/16, 31%), PIK3CA (3/16, 19%) and ERBB2 (3/16, 19%). In 3 patients with serial samples, 9 novel pathogenic alterations were detected in the second sample including ATM, CDKN2A, ARID1A, CCND1, CDK6, EGFR, PDGFRA, PIK3CA, and SMAD4. Conclusions: ctDNA alterations in patients with advanced penile cancer were frequently detected with similar prevalences to previously described tumor tissue analyses. New alterations observed on serial ctDNA assays may provide insights regarding tumor biology and inform drug development.