Abstract. Although plastid transformation has attractive advantages in plant biotechnology, it has been highly proficient only in tobacco. The lack of efficient semi lethal selection procedure along with the inefficient recombination through heterologous flanking regions used in transformation vectors prevented this technology to major crops. However, due to the published plastidial genomic sequences and the distinct features of their genomic parts lead the scientists to use of specific and new regions in transformation vectors. In this work, we worked out rrn23-rrn5 fragment as new flanking regions for Malvaceae, Caricaseae, Rutaceae and Solanaceae families by nucleotide homology search. Therefore, the PCR-amplified rrn23-rrn5 region was cloned into the cloning vector using competent cells of E.coli strains DH5α. The sequencing results showed the high homology compared to chloroplast genome of Gossypium sp. with identities of 99 % values.