Colorectal cancer (CRC) is driven by a complex spectrum of somatic mutations and structural variants that contribute to tumor heterogeneity and therapy resistance. In this study, we performed a comparative analysis of short-read Illumina and long-read Nanopore sequencing technologies across multiple CRC sample groups, encompassing diverse tissue morphologies. Our evaluation included general base-level metrics—such as nucleotide ratios, sequence match rates, and coverage—as well as variant calling performance, including variant allele frequency (VAF) distributions and pathogenic mutation detection rates. Focusing on clinically relevant genes (KRAS, BRAF, TP53, APC, PIK3CA, and others), we characterized platform-specific detection profiles and completed the ground truth validation of somatic KRAS and BRAF mutations. Structural variant (SV) analysis revealed Nanopore’s enhanced ability to resolve large and complex rearrangements, with consistently high precision across SV types, though recall varied by variant class and size. To enable direct comparison with the Illumina exome panel, we applied an exonic position reference file. To assess the impact of depth and PCR amplification, we completed an additional high-coverage Nanopore sequencing run. This analysis confirmed that PCR-free protocols preserve methylation signals more accurately, reinforcing Nanopore’s utility for integrated genomic and epigenomic profiling. Together, these findings underscore the complementary strengths of short- and long-read sequencing platforms in high-resolution cancer genomics, and we highlight the importance of coverage normalization, epigenetic fidelity, and rigorous benchmarking in variant discovery.