Three-line breeding strategy involving the use of WA-CMS lines is being extensively exploited for commercial cultivation of rice hybrids. Presence of an unedited 1.1-kb transcript of the mitochondrial orfB gene and its translated product leads to male sterility in rice. CMS based 3-line systems could enable breeders to use and recombine germplasm with fewer restrictions. Bringing the mitochondrial orfB gene into nuclear background may help to overcome such limitation. The male sterile lines possessing orfB gene in nuclear background were developed and characterized. Easy diagnostic tool is needed to identify the transgenic lines containing the orfB gene in order to study the inheritance pattern of the gene. In the present study, we developed PCR-based diagnostic tool for identifying nuclear male sterile lines with orfB gene.