Background: Penile cancer is a rare malignancy with limited genomic characterization in the litterature. Implementation of next-generation sequencing (NGS) enable detailed analysis of the cancer genome, providing opportunities to objectively categorize tumors based on biomarkers, which could guide diagnosis, prognosis, and treatment. The Genomic Medicine Sweden (GMS) GMS560 panel targets 560 cancer-associated genes and is commonly used in solid tumor analysis and has not been systematically evaluated for penile cancer. This study aims to assess the feasibility and performance of the GMS560 panel on formalin-fixed, paraffin-embedded (FFPE) penile cancer tissue, and to compare the three DNA library preparation kits, KAPA HyperPlus (Hyp), KAPA EvoPlus V2 (Evo), and Watchmaker DNA library prep kit with fragmentation (WM).