A seroepidemiological study of Brucella infections in multiple livestock species in the Borana pastoral system of Ethiopia was performed between December 2007 and October 2008. A cross-sectional multi-stage sampling technique was employed to select 575 cattle, 1073 camels and 1248 goats from the target populations. Sera were collected from the animals, and serially tested using Rose Bengal test and complement fixation test. Overall prevalence and prevalence with respect to explanatory variables were established, and potential risk factors for seropositivity were analysed using a multivariable logistic regression. The results showed that 8·0% (95% CI 6·0–10·6), 1·8% (95% CI 1·1–2·8) and 1·6% (95% CI 1·0–2·5) of the tested cattle, camels and goats, respectively, had antibodies to Brucella antigen. Positive reactors were found in 93·8% of the villages with more frequent detection of positive cattle (93·3%) than camels (56·3%) and goats (37·5%). Risk factors identified for cattle were: keeping more livestock species at household level (OR 4·1, 95% CI 1·9–8·9), increasing age of the animal (OR 2·8, 95% CI 1·3–6·0) and wet season (OR 3·3, 95% CI 1·6–6·9). Increase in household-level species composition (OR 4·1, 95% CI 1·2–14·2) and wet season (OR 3·7, 95% CI 1·5–9·1) were found to be risk factors for seropositivity in camels and goats, respectively. Existence of more than one seroreactor animal species in most villages and association of increased livestock species composition with seropositivity may add more credence to the possibility of cross-species transmission of Brucella infections. Although no attempt to isolate Brucella spp. was made, our results suggest that cattle are more likely maintenance hosts of Brucella abortus which has spread to goats and camels. This should be substantiated by further isolation and identification of Brucella organisms to trace the source of infection and transmission dynamics in various hosts kept under mixed conditions. In conclusion, the present study suggests the need for investigating a feasible control intervention and raising public awareness on prevention methods of human exposure to brucellosis.