Rabies is considered a deadly zoonotic disease that still has been causing huge fatalities in all over the world. The objective of the present study was to investigate the histopathological, immunohistochemical and molecular diagnosis of rabies in randomly selected ruminant’s brain tissue samples, slaughtered at eight abattoirs of Faisalabad, Pakistan. A total number of 192 brain samples of sheep (n¹=48), goats (n²=48), cows (n³=48) and buffaloes (n⁴=48) were procured and, immersed in 10% neutral buffer formalin for fixation purpose. Most of the samples were quite healthy and their percentage distribution of variable pathological lesions (neuronal necrosis, perivascular cuffing and vascular congestion) in all brain samples was noted, but found negative for rabies virus infection. However, one sample of goat and buffalo were diagnosed positive for rabies virus by exhibiting characteristic pathological lesions such as babe’s nodule, cavernous lesions, perivascular cuffing, satellitosis and degenerative changes in various sections of the brain. Similarly, immunohistochemistry showed intense characteristic antigen-antibody reaction by producing viral masses in cerebrum and cerebellum of the brain. Reverse transcriptase PCR (RT-PCR) further confirmed the findings by generating nucleic acid band against a standard DNA ladder. This study was the second insight into rabies cases scattered among domestic animals of Faisalabad. Proper awareness, reliable prophylactic measures and diagnostic methods must be established to stop the concurrent spread of the rabies virus in humans and animals.