Abstract The purpose of this study is to recognize the phenotypic behavior of cytoplasmic male-sterile (cms) line of leaf mustard synthesized by intra-specific hybridization and identify genetically transmitted molecular markers related to cytoplasm. Phenotypic variations of both vegetative and floral organs were observed in cms and its maintainer line. Pistil fertility was reduced correspondingly and recovered progressively with backcrossing. In BC 3, some flowers showed abnormality with pinnate, petaloidy, palm-like, carpelloidy or rudimentary silk-like stamens. The deformed floral phenotypes were also observed with closed or partially expanded corollas. By PCR-aided assay using specific primers from mitochondrial genes, atpA and one open reading frame (orf) were found to be distinctively present in the progenies of intra-specific F 1 hybrid, BC 2 and BC 3, which were proved to be maternally transmitted and genetically stable in various backcrossed progenies. The cms specific orf corresponds to a 663 bp-sized fragment with its own initiator (ATG) and terminator (TGA). Furthermore, it was deduced to encode for 220 amino acids, named orf220 thereafter, and was shown to belong to the plant mitochondrial membrane YMF19-like protein family. Transcript of orf was exclusively present in cms and absent in its maintainer. Moreover, atpA gene was expressed in a larger transcript size in BC 3 progeny than in the maintainer.