With the invention of PCR, knowledge of evolution of DNA, particularly of fish and phylogenetic relationships of fish has increased rapidly. This enzymatic cloning technique, together with direct sequencing has simplified and dramatically accelerated the accumulation of DNA sequence information. rDNA (ribosomal DNA) are organized as multiple copies of a repeated unit consists of transcribed zone with coding regions for the 18S, 5.8S and 28S RNA genes, internal external transcribed spacers and surrounded by non-transcribed spacer sequences. In present work, the DNA of Clarias batrachus was amplified with specific primers and observed for molecular, genetic characterization and phylogenetic analysis. The evolutionary relationship of C. batrachus was established by phylogenetic tree. The overall base size of sequence of various fragments of 45S ribosomal DNA that contain 18S, ITS1, 5.8S, and 28S coding region is 5020 bp long. The results obtained in these aspects enrich the knowledge of C. batrachus at molecular level and helps in public database stock. These new sequences will contribute to a better understanding of the patterns of evolution of this scarcely studied genus.