Abstract: Direct repeat sequences widely exist in eukaryotic and prokaryotic genomes, which involves in human diseases like hereditary neuromuscular neurodegenerative disease. Thus, it is of great importance to quantify the frequency of the deletion of direct repeat sequences. Using the high-throughput microscopy imaging and analysis technology, we designed a three-color fluorescent-based reporter system to quantify the frequency of the deletion of direct repeat sequences. We found that the frequency of the deletion of direct repeat sequences decreased in the recA mutant in P. aeruginosa, and the absence of RadA and UvrD increased the occurrence frequency of the deletion of direct repeat sequences, but the frequency of the deletion of direct repeat sequences was independent of the following factors: fitness or promoter variants etc. This work can be helpful to the further understanding of the deletion of direct repeat sequences, which also provide a novel method to quantify the deletion of direct repeat sequences.