HCT116 is a colorectal cancer cell line frequently used in anti-tumor drug development experiments as well as in studies of the molecular machinery of eukaryotic cells. It is well characterized by the presence of several single-nucleotide and short mutations in multiple oncogenes and tumor suppressor genes, including KRAS, PIK3CA, MLH1, CTNNB1, CDKN2A, TGFBR2, and BRCA2. However, its landscape of large genomic rearrangements (LGRs) and copy number variants (CNVs) is still far from being fully understood. Therefore, the aim of this study was to identify LGRs and CNVs in several HCT116 cell line samples using Oxford Nanopore sequencing technology, including three samples from the SRA NCBI database, and to compare common and unique variants across all samples. Using the recently developed eLaRodON tool, we identified 22,666 common LGRs, among which more than 70% of tandem duplications and deletions larger than 80 kb were confirmed by CNV analysis. Among LGRs affecting protein-coding sequences, two in-frame rearrangements were identified: a deletion of exons 4–6 and a duplication of exon 10 in the CCSER1 gene, which encodes a cell division regulator protein. Given its high rearrangement rate in various tumors and the clinical significance of its overexpression, this finding may be potentially useful in future research on this cell line. Regarding differences between samples, we found that LGRs in the laboratory sample and in one of the three SRA NCBI samples occurred more frequently via ALR/Alpha repeats than via Alu repeats, in contrast to common LGRs and those unique to the other samples, a finding that may indicate the presence of unique mechanisms of genomic instability. Thus, this study reveals a broad spectrum of large genomic rearrangements and copy number variants that can be identified in the HCT116 cell line using Oxford Nanopore sequencing, including rearrangements specific to distinct cell line samples.