Cell line models are essential tools to study the molecular mechanisms underlying tumor initiation and progression. There are limited treatment options for penile squamous cell carcinoma (PSCC), accounting for 1-2% of male tumors in developing countries, and limited progress in preclinical research in PSCC due to lacking available models with identified genomic characteristics. Here, biological and molecular characteristics and whole-genomic alterations were analyzed in a panel of PSCC cell lines newly established in our laboratory. These cell lines were all human papillomavirus (HPV)-negative, epithelial-like, immortalized, and tumorigenic in nude mice, whereas they displayed different proliferation, migration and invasion capacities in vitro, and tumorigenic ability in nude mice. They were all cisplatin sensitive, anti-EGFR therapy resistant, and androgen irresponsive. Whole-genomic sequecing analysis revealed that transition mutations (C:G>T:A and T:A>C:G) were the most common substitution types in these cell lines, whereas ERCC5, TP53, PTH1, CLTCL1, NOTCH2, MAP2K3, CDK11A/B, USP6, ADCH5, BCLAF1, CDKN2A, FANCD2, HRAS, and NOTCH1 were the most frequently altered genes. Amplifications of MYC, PLAG1, NCOA2, RUNX1T1, COX6C, and EGFR and losses of FBXW7, TET2, XPC, and FANCE were frequently observed in cell lines. The exomic variations between cell lines and their corresponding cancer tissues were highly consistent. Genetic variations were mainly involved in the MAPK, Jak-STAT, TGF-beta, Notch, and apoptosis signaling pathways. Conclusively, these panel of PSCC cell lines established in our laboratory harbor some common or specific biological characteristics and genomic variations, and they may serve as optimal models to investigate the molecular mechanisms underlying the progression, metastasis, relapses, and treatment resistance of PSCC and to develop effective treatment strategy.