Cytoplasmic male sterility (CMS) system is most important genetic mechanism for affordable and genetically homogenous hybrid seed production. The present study was aimed to evaluate the CMS lines and their maintainers for floral traits as well as assess suitability of mitochondrial DNA (mtDNA) markers to distinguish the fertile/sterile plants at seedling stage. Morphological observations revealed that introgression of CMS trait significantly reduced flower size (length, diameter), petal size (length, width), stigma and anther length. Out of five mtDNA markers, namely Orf138, orf222-orf224, atp6-orf224, Orf138 and BnTR4, two markers (Orf138 and orf222-orf224) exhibited dominant pattern and clearly distinguished CMS lines from their fertile maintainers. However, atp6-orf 224 and BnTR4 showed polymorphic amplicons of different size between them. These new CMS lines have great potential in hybrid breeding and hybrid seed production in Indian cauliflower. The two mtDNA markers (Orf138 and orf222-orf224) will be useful for early detection of Ogura CMS system during introgression programme and for clarification of doubtful seeds.