Mitochondrial DNA (mtDNA) ribosomal genes have been used as molecular markers for fish species identification in many studies. In this study, two mtDNA ribosomal genes namely 12S rRNA and 16S rRNA have been utilized to characterize ornamental fish species from Poeciliidae family. Five ornamental fish species namely Poecilia sphenops, Poecilia reticulata, Limia vittata, Xiphophorus hellerii and Xiphophorus maculatus were used. PCR amplification was performed and DNA sequencing was carried out in order to study the genetic relationship of fish species. More polymorphisms were seen in 16S rRNA as compared to 12S rRNA. Phylogenetic tree results showed two clusters with one cluster consisting of Limia vittata, Xiphophorus hellerii and Xiphophorus maculatus and the second cluster consisting of Poecilia sphenops and Poecilia reticulata.