Background: The species of liver fluke of the genus Fasciola are obligatory parasites that inhabit the biliary ducts of herbivorous animals as well as human. Understanding genetic structure and status of genetic variation of F. hepatica populations has important implications for epidemiology and effective control of fasciolosis. Aim: To genetically characterize Fasciola isolates from different hosts from Assiut, Egypt using sequence analysis of the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA). Methods: Three adults F. hepatica were isolated from naturally infected sheep and fragments of Fasciola spp. were extracted from three human cases by ERCP (Endscopic Retrograde Cholangio-Pancreatography). Genomic DNA was extracted from preserved flukes. Conventional polymerase chain reaction (PCR) with a set of arbitrary primers was used to estimate genetic variation within the species. Ribosomal ITS-1 and ITS-2 regions of the isolates were amplified. The amplicons were sequenced at ITS-1 and ITS-2. Results: Both regions were amplified successfully for all samples and bands ranged between 400 bp and 650 bp were shown in all cases. Comparison of the obtained ITS sequences to those from known Fasciola species circulating globally and retrieved from GenBank revealed that the present worms were genetically identical (100%) with F. hepatica. Different isolates did not show any significant genetic variations in rDNA-ITS-1 and ITS-2 as all the sequences showed to be 100% identical. Conclusions: These findings have implications for studying the population genetics, epidemiology, diagnosis and control of fasciolosis especially in human.