Analysis of RAPD (Randomly Amplification Polymorphic DNA) was performed between cytoplasmic male sterility line and its maintainer line of cauliflower. Totally 2160 detectable bands were obtained by RAPD using 406 10-bp random primers. Averagely, 5 to 10 bands were produced per primer. Among all the primers only the amplification of primer S2121 was polymorphic in two lines. A 934-bp specific band was only detected in maintainer line. After cloning and sequencing, specific primers were designed to transform the RAPD marker to PCR marker, which was named S2121(900). To identify the specificity of S2121(900), southern dot blotting was performed. To further identify its specificity, individual plant and candidate materials testing were also performed. All these results indicated that the S2121(900) was specific. It can be used to screen the maintainer lines of cauliflower in early stage. Analysis of the sequence suggested that this fragment was high homologous with the part sequences of mitochondrial genome in Brassica napus and Arabidopsis thaliana. So we supposed the S2121(900) may also derive from mitochondrial genome. Our results here offer new clues for explaining the molecular mechanism of cytoplasic male sterility of cauliflower in other way.