DNA topoisomerase I (topo I) is the principle target for Camptothecin and its analogues. The topo I gene is located on chromosome 20q11.2-q13.1 and variation in topo I gene copy number has been shown to have impact on the in vitro sensitivity to topoisomerase I inhibitor chemotherapy. Fluorescence in situ hybridization (FISH) was used to detect and compare the TOPO I gene copy number between metaphase and interphase nuclei in a panel of 7 colorectal cancer cell lines. TOPO I gene copy number varied from 2 to 8 between cell lines, and signal in interphase nuclei demonstrated a linear relationship with that detected in metaphase nuclei. The structure of gene amplification included isochromosome formation, amplicon extension, and marker chromosome generation. Comparative genomic hybridization (CGH) was then used to further define the region of gain on chromosome 20. The region of gain contained the topo I gene and involved nearly all of 20q in most cases. This demonstrates a high degree of intrinsic variation in topo I gene copy number and the involvement of a 20q amplicon in colorectal cancer, which may have important implications for colorectal tumorigenesis and the use of chemotherapy.