Objective To establish a method for genetic detection of pigeon microsatellite DNA in a closed-groupexperiment. Methods Sixty-one pigeon microsatellite loci were selected and designed through literature search and SSR(simple sequence repeats) Hunter software screening. PCR amplification and condition optimization were performed usingpigeon genome samples. Twenty microsatellite loci with abundant alleles and distinct amplification bands were obtainedusing agarose gel electrophoresis. Through comparative analysis of STR (short tandem repeat) scanning, 16 loci suitable fortesting the genetic quality of experimental pigeons were selected and applied to two closed-group pigeon populations.Results Sixteen microsatellite DNA loci were selected to establish the genetic detection method of a closed group ofpigeons. Conclusions A genetic detection method based on microsatellite DNA is established for a closed group of pigeons. This method lays a foundation for genetic quality control in a closed-group pigeon population.