Objective To establish a high throughput general multiple competitive polymerase chain reaction (cPCR) detecting method of copy number variations (CNVs) for the population of chromosome 1 substitution strains from wild mice. Method The selected 14 loci, including 11 CNVs on chromosome 1 and internal control loci on other three chromosmes (Chr 7, Chr 19 and Chr X), were detected based on the universal fluorescent primer multiple competitive polymerase chain reaction. All specific cloned plasmids were constructed as competitors. Results Altogether 11 CNVs were designed in one panel, and the copy of Chr X accurately reflects the gender. Conclusions A rapid and high-throughput fluorescent multiplex cPCR assay is established which can be used for detection of copy number variations on chromosome 1 in mice.