The aim of this study is to estimate the prevalence of Toxoplasma gondii infection in 527 sheep from 4 governorates of Tunisia by Elisa (350 animals) and PCR (177 animals). The seroprevalence in sheep was estimated to be 1.8% (N = 166) in the governorate of Siliana (North Tunisia) and 19% (N = 184) in the governorate of Kasserine (Central Tunisia) with a commercial Elisa kit. T. gondii DNA was extracted from the apex of the heart in 25.5% (N = 106) of sheep from the Sidi-Bouzid governorate (Central Tunisia) and 12.7% (N = 71) from the Ben-Arous governorate (North Tunisia). There was no statistically significant difference between different age categories' prevalence within each locality. Our results indicate that T. gondii infection is frequent in Tunisian sheep. The implementation of a national control programme against toxoplasmosis should not neglect sheep as a frequently infected intermediate host.