Abstract: Abstract: 【Objective】Two kinds mitochondrial genome BAC library were constructed using JA cytoplasmic male sterile in cotton and its maintainer lines. 【Method】According to many methods of crops, a high-efficient technology system of BAC library construction was established. This system contains that isolation of mitochondrion, preparation of high-molecular weight（HMW）mtDNA, partial digestion of megabase DNA with a restriction enzyme, selection of digested fragment and transformation with high efficiency. We designed a set of primers based on the conserved sequences of the orfB、coxⅠ and nad4L coding region in other plants. These genes locus were subjected to PCR analysis using these primers. The PCR products using mitochondrial cotton DNA as a template were further sequenced after cloning.【Result】Two kinds mitochondrial genome BAC library were constructed using JA cytoplasmic male sterile in cotton and its maintainer lines. Each library consisted of about 2600 clones with average insert size 22.29kb(or 21.36kb), ranging from 10.3kb to 37.5kb. Three genes were colned from mitochondrial genome of cotton, and It was testified that orfB, coxⅠ and nad4L were conversed between Jin-A cytoplasmic male sterile and its maintainer lines. With mitochondrial genes orfB, coxⅠ and nad4L as probes, We obtained all positive clones from two BAC library by colony in situ hybridization.【Conclusion】It was calculated that each library contains approximate 79 and 76 times of cotton mitochondrial genome and provide 100% possibility to isolate any interested cotton genes or sequences in each library.