Paratuberculosis (PTB) in buffaloes is a chronic enteric disease triggering health implications and huge economic losses in livestock. This study was designed to explore a simple, cost-effective diagnostic approach for PTB in water buffaloes. Blood (5 ml/animal) and intestinal tissue samples accompanied by lymph nodes associated with mesentery were collected from weak and diarrhoeac animals slaughtered at local abattoirs. Out of total n=771 clinically suspected animals, only n=53 carcasses were sampled based on gross observation. Tissue smears of the gut mucosae were obtained and were made adopting special staining protocol. Tissue samples were processed by paraffin sectioning and stained with Ziehl-Neelsen and Hematoxylin-Eosin staining methods. Acid-fast bacilli were observed only in 11/53 cases on mucosal tissue smears. Pressure atrophy of small intestine villi were evident, and the mucosae were found sloughed off. The submucosae were heavily infiltrated with mononuclear cells and multifocal cellular nodules dominated by epithelioid macrophages. The foamy cytoplasm of the macrophages appeared to be engorged with acid fast bacilli and depicted the positive cases. All tissue sections of the suspected samples showed 100 percent +ve results while only 20.8 percent samples were found +ve with smear method. All histo-pathologically positive cases were further confirmed by ELISA based serological analysis. Therefore, it was concluded that histopathology is an economical and yet the most trusted tool for diagnosing bubalian PTB in countries like Pakistan.