To develop the.ne and reliable molecular markers in Arabidopsis thaliana, this work analyzed the whole ge-nome sequence of Columbia and Landsberg erecta with comparative genomics. From total 10 449 insertion/deletion (IN-DEL) DNA fragments that have no less than six bases, we screened 2 321 INDEL molecular markers. For each marker, at least one pair of primers (a total of 4 764) were designed. Based on this, we randomly selected 20.rst-pass mapping molecular markers that are relatively evenly distributed in.ve chromosomes of Arabidopsis thaliana. When using these molecu-lar markers, we were able to obtain stable polymerase chain reaction (PCR) and agarose gel electrophoresis results under uni-form reaction system and ampli.cation conditions. This study will facilitate researchers to.nd suitable molecular markers, and under the common instrument conditions and lower experimental costs, to quickly locate the mutant gene and improve the e.ciency of gene map-based cloning.