The present study aims to determine the morphological features of amphistome adults and eggs combined with examining pathological, hematological and bile biochemical response in buffaloes to Gigantocotyle explanatum infection. Of the 558 slaughtered buffaloes from central Punjab, Pakistan, 18.99% were infected, and morphometric analyses were made of 115 mature amphistomes and gravid eggs with traditional microscopic methods on the basis of standardized measurements. Amphistomes were flattened and stained in Borax carmine, some were sectioned in the median sagittal plane and histological slides of the worms were prepared for detailed morphological studies. Bile tissues, aspirates and blood samples of the same animals were analysed. The infection was found prevalent throughout the year but during post monsoon season it was relatively high. Morphological identification of G. expalanatum was carried out on the basis of size and shape of worm. The hematological and bile biochemical changes were found in two groups. Pathologically significant changes were observed in the infected tissue. The parasites had formed pocked in the bile duct mucosa, glandular hyperplasia, thickened blood vessels. The results demonstrate that morphological markers, pathological, hematological and bile biochemical studies are effective tools that could be used as complementary in diagnosis of amphistome infections in ruminants.