¹Department of Animal Sciences, Faculty of Biological Sciences, Quaid-i-Azam University Islamabad, 45320, PAKISTAN DOI : 10.9775/kvfd.2019.22777 The family Paramphistomidae including Gigantocotyle explanatum regularly infects ruminants and causes immense economic losses to the livestock industry by decreasing dairy products and growth rates. The present study was aimed to determine the novel molecular data of G. explanatum in Pakistan using ribosomal DNA (ITS1-5.8S, ITS2) regions. Adult flukes, G. explanatum, were collected from bile ducts of infected buffaloes. The most relevant sequences from the other parts of the world were downloaded from the GenBank. High intraspecific variations were obtained at 5 end region of ITS1 gene. The 3 end of ITS1 was conserved and showed 96% similarity with Paramphistomum cervi (KJ459936). The nucleotide blast search of 5.8S gene revealed that 40 sequences from trematodes had 98% to 99% identity with present sequence and found genetically identical to P. cervi (KJ459938) and Dicrocoelium chinensis (KF734784) from China. The ITS2 gene of investigated isolates showed no variation with Myanmar (AB743577), while blast search revealed 96-100% similarity with isolates from Myanmar, India, Bangladesh and China. This study demonstrates the utility of ITS2 and 3´ end ITS1 sequences as a valuable tool for elucidating species phylogenetic relationship in south Asia. This sequencing data will facilitate more accurate identification of G. explanatum, enabling future work to resolve many ambiguities in the literature regarding this species. Keywords : Gigantocotyle explanatum; Water buffaloes, Pakistan, Genetic characterization