In present study, 50 samples representing Fasciola (Platyhelminthes: Trematoda: Digenea) from cattle and buffaloes of Pothwar region were characterized genetically by sequences of the first (ITS-1) and second (ITS-2) internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). The ITS rDNA was amplified from individual liver flukes by polymerase chain reaction (PCR), and the amplicons were sequenced directly. The lengths of the FG ITS-1 and ITS-2 sequences were 391 and 329 bp, respectively, for all Fasciola gigantica samples sequenced. For Fasciola hepatica samples sequenced, FH ITS-1 was not amplified and length for ITS-2 sequences was 330 bp. Molecular identification using rDNA internal transcribed spacer 1 and 2 characterizes Fasciola   spp. in Pothwar to be an intermediate/ hybrid resembling more with F. gigantica. The results also indicated that Fasciola spp in cattle and buffalo are genetically similar, as both hosts' possess Fasciola with the same number of base pairs in their internal transcribed spacers as proved by gel electrophoresis and sequencing. To determine the phylogenic location of Fasciola spp. of Pakistan based on rDNA molecular data, ribosomal ITS regions were sequenced and compared with other fasciolid species from NCBI databases for sequence homology analysis using BLAST and ClustalW programs. The phylogenetic trees revealed a close relationship with isolates of F. gigantica from China, India, Iran and Vietnam. This is the first demonstration of the existence of hybrid or an intermediate Fasciola species in Pakistan by a genetic approach, which provides foundation for further studies in different agro ecological zones of Pakistan.

Key words: Fasciolosis, Intermediate Fasciola, Molecular characterization, Pothwar region