Background Penile squamous cell carcinoma (PSCC) is a rare, aggressive malignancy with a high risk of mortality due to metastasis. A comprehensive understanding of its mutational landscape is critical for improving early detection and therapeutic strategies. Methods We analyzed tissue samples from 28 patients with PSCC treated at the Cancer Hospital of the Chinese Academy of Medical Sciences between January 2019 and March 2023. DNA from primary tumors and/or matched inguinal lymph nodes underwent targeted sequencing. Somatic mutations were profiled to compare primary and metastatic lesions. Statistical analyses assessed associations between mutational features, clinical outcomes, and treatment responses. Results A total of 980 mutations were identified across 354 genes. Frequently mutated genes included TP53 (67.5%), TERT (45%), CDKN2A (40%), FAT1 (37.5%), and NOTCH1 (30%). Copy number variations (CNVs) revealed amplifications in EGFR, SOX2, and MSH6 and deletions in CDKN2B and CDKN2A. A strong correlation was observed between mutational profiles of primary and metastatic lesions (r = 0.61, p < 0.001). Metastatic tumors exhibited higher tumor mutational burden (TMB) than primary tumors (38.9% vs. 9.5%, p = 0.030) and displayed a greater prevalence of mismatch repair deficiency-associated mutational signatures. Patients with higher TP53 mutation frequencies responded more favorably to immune checkpoint inhibitors (p = 0.024), with treatment efficacy strongly correlated (AUC = 0.938). Conclusion Key mutational alterations in PSCC, including high TMB and TP53 mutations, have significant implications for early diagnosis and personalized therapies. These findings support the potential use of specific genetic markers to guide targeted therapeutic approaches. Micro-Abstract This study profiles somatic mutations in penile squamous cell carcinoma (PSCC) using targeted sequencing of 28 patients. Key mutations in TP53, TERT, and CDKN2A, alongside higher tumor mutational burden in metastatic lesions, highlight the potential for using genetic markers in early diagnosis and personalized therapies, including immune checkpoint inhibitors.