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Abstract: Recovering the genetic divergence between species is one of the major interests in the evolutionary biology. It requires accurate estimation of the neutral substitution rates. Arabidopsis thaliana, the first whole-genome sequenced plant, and its out-crossing relatives provide an ideal model for examining the split between sister species. In the study, rates of molecular evolution at markers frequently used for systematics and population genetics, including 14 nuclear genes spanning most chromosomes, three noncoding regions of chloroplast genome, and one intron of mitochondrial genome, between A. thaliana and four relatives were estimated. No deviation from neutrality was detected in the genes examined. Based on the known divergence between A. thaliana and its sisters about 8.0–17.6 MYA, evolutionary rates of the eighteen genes were estimated. Accordingly, the ratio of rates of synonymous substitutions among mitochondrial, chloroplast and nuclear genes was calculated with an average and 95% confidence interval of 1 (0.25–1.75): 15.77 (7.48–114.09): 74.79 (36.27–534.61). Molecular evolutionary rates of nuclear genes varied, with a range of 0.383–0.856×10−8 for synonymous substitutions per site per year and 0.036–0.081×10−9 for nonsynonymous substitutions per site per year. Compared with orthologs in Populus, a long life-span tree, genes in Arabidopsis evolved faster in an order of magnitude at the gene level, agreeing with a generation time hypothesis. The estimated substitution rates of these genes can be used as a reference for molecular dating. [Copyright &y& Elsevier]