The genetic variabilities in internal transcribed spacers ITS1 and ITS2 of rDNA are explored to a limited extent in vertebrates including fish species. This study explores unique molecular signature in these spacers from multiple catfish species from the order siluriformes. Supporting the concerted evolution, both ITS1 and ITS2 were found prevalent with microsatellite and random repeats, with diverse distribution in each species in terms of nucleotide composition, their position and length, giving a species-specific impression in repeats. Their secondary structures too have shown distinct structural homology with variations in helix lengths, loops, and non-canonical base pairs, where ITS2 4-helix carries additional nine conserved motifs while that of ITS1 was found highly branched with multiple sub-branching unique to each species and a long tail helix common in all. This combination of secondary structures and repeats have formed unique molecular signatures distinguishing each catfish species. Since the evolutionary and maturation biology of rDNA is similar in vertebrates, these molecular signatures in internal spacers could effectively contribute to the molecular characterization across the diverse group of catfish species.