Detection of Chromosomal Abnormalities (CA) is important for cancer patients. Compared to common cancer cytogenetics methods including karyotype, fluorescent in situ hybridization (FISH), and chromosomal microarray (CMA), optical genome mapping (OGM) is an innovative method to detect CA. The objective of this pilot study was to determine a stepwise approach to adopt OGM in the clinical setting. A blinded study was used to compare OGM, karyotype, FISH, and CMA on five cancer cases (four leukemia/lymphoma and one solid tumor). Next-generation sequencing and a gene fusion assay were also used for confirmation. We used a stepwise approach to analyze OGM calls. Three-quarters of 406 OGM calls in this study were filtered out according to genomic sizes, cancer genes, and knowledge based cancer databases. The remaining 89 cells were analyzed independently/blindly to determine their reportability/ pathogenicity. OGM revealed all chromosome abnormalities previously identified by karyotype and FISH (a total of 98 loci) and achieved a sensitivity of 100%. Compared to CMA, the sensitivity of OGM was 88% (including copy-number neutral loss of heterozygosity (CN-LOH)) and 96% (excluding CN-LOH). While, OGM cannot detect CN-LOH, while it revealed 71 additional calls, 52% of breakpoints involved genes and 7.7% of breakpoints involved known cancer genes. Although these additional/unrecognized calls may play roles in cancer development and formation, they are currently variants of unknown clinical significance. OGM effectively characterized a complex rearrangement and a chromothripsis-like event. OGM is a reliable method for detecting both copy number variants and structural variants in cancer genomes using a stepwise approach and can be adopted smoothly into the clinical setting as a promising alternative to routine cytogenetic techniques of karyotype/FISH/CMA assay.