The aim of the study was to determine the burden of Toxoplasma gondii-infections in sheep in Sicily, southern Italy and the risk factors for infection. Sera from 1961 sheep were collected just before slaughtering from 62 farms located in 8 out of 9 Sicilian administrative districts. The sera were analysed for Toxoplasma-specific IgG antibodies using commercially available enzyme-linked immunosorbent assay. Sheep less than 4 weeks old were further analysed by ELISA for Toxoplasma-specific IgM-antibodies. Data on farm size and location were obtained from slaughterhouse sanitary reports and through structured telephone interviews of the veterinary officers from public health districts. The overall seroprevalence of Toxoplasma-specific IgG-antibodies were 49.9% (937/1876) by ELISA. Eighty-seven (54/62) percent of the farms had at least one Toxoplasma-positive animal. All the farms fed the animals outdoor on pasture and only one was claiming organic farming. Having cats on the farm, age of the animals, farm size and the use of surface water sources for drinking were all significantly associated with T. gondii-infected animals on the farm. T. gondii infection in mutton used for human consumption is very prevalent, and eating unprocessed sheep and lamb meat has a high risk of transmitting infections to humans. The presence of cats on the farm, farm size and using surface water as drinking water for the animals were risk factors for infection in sheep, with age as a significant confounder.