Purpose: Primary urethral cancer (PUC) is an uncommon malignancy with scarce diagnostic and treatment options, resulting in a limited understanding of its genetic foundation. This exploratory study compares gene expression profiles between urethral cancer and histologically normal urethral tissue from penile cancer patients (HN-PC). Materials and Methods: Twenty-three urethral specimens (13 malignant and 10 HN-PC) were collected between 2015 and 2023. RNA was isolated and analyzed via bulk RNA sequencing. Differentially expressed genes were identified, and multiple enrichment analysis techniques were performed including gene set enrichment analysis (GSEA), gene ontology analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Results: A total of 1,212 significantly differentially expressed genes (false discovery rate [FDR] <0.01) were recognized with strong differentiation between the 2 cohorts. Twenty-two GSEA gene sets were identified as significantly enriched (FDR <0.01) with 392 significantly upregulated (FDR <0.01 and log2 fold change >1) genes within the leading edges. Gene ontology analysis highlighted chromosome organization, cell cycle regulation/processes, nuclear division, and tissue development. KEGG analysis revealed similar findings with the addition of enhanced viral protein interactions. Conclusion Next-generation sequencing revealed several genes and pathways commonly altered in PUC and also offered a set of new targets for future diagnostic and therapeutic trials.