Chinese hamster ovary (CHO) cell lines are widely used for scientific research and biotechnology. A CHO genomic bacterial artificial chromosome (BAC) library was constructed from a mouse dihydrofolate reductase (DHFR) gene-amplified CHO DR1000L-4N cell line for genome-wide analysis of CHO cell lines. The CHO BAC library consisted of 122,281 clones and was expected to cover the entire CHO genome five times. A CHO chromosomal map was constructed by fluorescence in situ hybridization (FISH) imaging using BAC clones as hybridization probes (BAC-FISH). Thirteen BAC-FISH marker clones were necessary to identify all the 20 individual chromosomes in a DHFR-deficient CHO DG44 cell line because of the aneuploidy of the cell line. To determine the genomic structure of the exogenous Dhfr amplicon, a 165-kb DNA region containing exogenous Dhfr was cloned from the BAC library using high-density replica (HDR) filters and Southern blot analysis. The nucleotide sequence analysis revealed a novel genomic structure in which the vector sequence containing Dhfr was sandwiched by long inverted sequences of the CHO genome.