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A total of 956 faecal samples were collected from buffaloes representing 21 districts of different agro-climatic zones of the Punjab state, India during April, 2013 to May, 2014 for detection of various gastrointestinal helminth (GIH) parasites. Coprological examination of the samples revealed an overall prevalence of 43.20% for GIH with mean egg per gram of faeces ranging from 170±7.07 to 226±7.14. The parasites recorded with their respective prevalences (%) in the descending order were: strongyles (27.62%), amphistomes (16.94%), Fasciola spp. (5.23%), Moniezia spp. (4.18%), Trichuris spp. (2.2%), Strongyloides spp. (1.88%) and Capillaria spp. (0.10%). The results of multivariate analysis showed that prevalence of GIH in buffalo population was associated with various risk factors viz. districts (P=0.000; OR: 0.474; CI 95%: 0.182-1.234), agroclimatic zones (P=0.000; OR: 1.156; CI 95%: 0.64-2.082) and seasons (P=0.316; OR: 1.138; CI 95%: 0.838-1.545). The maximum prevalence of GIH infection was recorded in monsoon season (47.84%) and lowest in winters (39.76%) with non significant differences (P>0.05). Agroclimatic zone-wise prevalence of GIH revealed the highest prevalence in undulating plain zone receiving maximum annual rainfall (62.88%) and lowest in western plain zone with minimum annual rainfall (33.33%) and the differences were highly significant (P<0.01). Coproculture analysis of the strongyle positive samples revealed the presence of larvae of genera Oesophagostomum, Haemonchus, Trichostrongylus, Cooperia, Chabertia and Strongyloides in decreasing order of their prevalence. The data generated could be of immense help in formulation of effective strategies for gastrointestinal parasite control in different agro-climatic zones of Punjab.