Abstract: Present study tends to investigate the existence of the bacterial oncogenes- rol genes of Agrobacterium rhizogenes Riker., in Nicotiana rustica, N. plumbaginifolia and their hybrid. These Nicotiana species and hybrid were normal morphologically under field conditions without any neoplastic growth. So, the habituation studies were carried out to identify the presence of rol genes by culturing the leaf discs on hormone supplemented Murashige and Skoog medium and then shifting to MS basal medium. Habituation showed phytohormone independent growth response even after shifting to MS basal medium indicating the presence of rol genes. Then, Polymerase Chain Reaction analysis showed amplification of rol A, B and C genes in N. rustica, rol A and C in N. plumbaginifolia and the hybrid. The amplified genes were sequenced and deposited in GenBank. The homology between the novel genes was compared with the rol genes of A. rhizogenes as well as the other plants species containing rol gene homologues using Pairwise sequence alignment search tool. Results were discussed in terms of the occurrence of bacterial genes in plants, the extent of their expression and the probable reasons for silencing of the foreign genes.