Abstract: The abnormal mitochondrial gene may lead to tobacco cytoplasmic male sterility. sdh3 and sdh4 are important genes coding subunits of mitochondrial complex II. To study the relationship between mitochondrial genes sdh3, sdh4 and cytoplasmic male sterility, the mitochondrial DNA from 3 male sterile lines and their maintainer lines of Nicotiana tabacum cultivars were extracted, specific primers were designed according to the complete nucleotide sequence of the tobacco mitochondrial genome in GenBank to amplify purpose genes from tobacco mitochondrial DNA. PCR products were cloned and sequenced. The sdh3 and sdh4 nucleotides and encoded protein structures were analyzed with bioinformatics to find differences between male sterile lines and maintainer lines. The results showed that the 28th base of sdh4 of male sterile lines gene changed. The sdh3 of male sterile lines gene lost 10 bases, and lead to the encoded protein altered, this is much more likely to be the key factors interference with the function of mitochondrial succinic dehydrogenase subunit 3, and one of root factors generated tobacco CMS.