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Background: The rapid development of sequencing technologies have been accumulated amounts of genomic data, which provide a powerful opportunity for revealing the origin of life.With the accomplishments of different genome projects, more than one chromosome is found in some prokaryotic genomes, while much fewer researches on the evolution of the diverse chromosomes haven been reported.Therefore, the evolutionary relationships of chromosomes in prokaryotic genome are studied based on codon bias analysis, which provide theoretical support for the related research in the future.