Molecular characterization and assessment of genetic variation in 28S region especially divergent domain D9 and D11 ribosomal DNA in fresh water catfish Ompok pabda, O. pabo, O. bimaculatus and Wallago attu was carried out. The nucleotide sequences and GC% of divergent domains of 28S ranged from 419-427 bp and 61% in D9 region and 686-688 bp and 55% in D11 region. In D9 region, O. pabda and O. bimaculatus were 100% similar, whereas other species were found 99% similar. In D11 region, O. pabda with O. pabo and O. bimaculatus were 100% similar and other species were 99% similar. After sequence analysis, Ompok species exhibited more variability from W. attu. DNA sequence data play an essential role in the reconstruction of evolutionary relationships among the organisms, resulting in insights in genetic affinities that may confirm or conflict with traditional taxonomy because of attractive properties, the rDNA is popular for examine phylogenetic relationships and for studying genetic variability and divergence within and between species.