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A serolocigical survey was conducted for the detection of Theileria parva antibodies in 176 African buffaloes (Syncerus caffer) sampled between 1996 and 2005 in livestock-wildlife interface areas of Zambia. Rhipicephalus appendiculatus, Rhipicephalus species, and Amblyomma variegatum were the most abundant tick species identified on buffaloes. T. parva sero-positives were reported in buffaloes sampled from game management areas at Mlanga and Nanzhila bordering the Kafue National Parks and in the Lochnivar National Park while buffaloes sampled from Lower Zambezi National Park were sero-negative. Given that Game Management Areas serve as interface areas that permit the co-existence of livestock and wildlife in similar ecological habitats our findings suggest that buffaloes could play a significant role in the epidemiology of theileriosis in livestock-wildlife interface areas. Thus far, the disease has only been reported in livestock and is herein being reported in the African buffalo for the first time in Zambia.