Fascioliasis is an important parasitic zoonosis with an estimated annual economic loss at more than US$3 billion in animals and infection risk in more than 180 million people worldwide. Given the economic and public health significance of this trematode, a periodical evaluation of its prevalence and the associated risk factors as well as the evolutionary relatedness is needed. Here, 1560 cattle and 1630 goat livers were examined for the presence of adult Fasciola among animals slaughtered in slaughterhouses of at Assiut and Sohag Governorates, Upper Egypt. Molecular characterization and phylogenetic relationships were determined based on the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA (rDNA). The overall prevalence of fasciolosis was based on morphological identification of the recovered Fasciola adult worms and the prevalence was 5.1% and 3.5% among cattle and goats, respectively. Though the age, gender, and housing system significantly affected the disease in cattle, none of these variables had a significant impact on goat fasciolosis. Nonetheless, the sampled goats were found at increased risk of being infected with Fasciola hepatica as the age increases and when the winter season emerges. Phylogenetic analyses showed that both Fasciola gigantica and Fasciola hepatica sequenced in this study had a common ancestor and they fall in one clade with several previously reported Egyptian and worldwide fasciolae from animals and humans. This study demonstrates that the disease is currently circulating among animals in Assiut and Sohag Governorates that warrants the urgent need for application of the appropriate control measures as well as evaluating the possible ongoing zoonotic burden in people residing such areas.