Toxoplasma gondii seroprevalence was determined in 3333 sheep sera from 125 distinct sheep flocks in Scotland, with the majority of flocks being represented by 27 samples, which were collected between July 2006 and August 2008. The selected farms give a representative sample of 14,400 sheep holdings identified in the Scottish Government census data from 2004. Overall T. gondii seroprevalence, at individual sheep level, was determined to be 56.6%; each flock tested, had at least a single positive animal and in four flocks all ewes tested positive. The seroprevalence of sheep increased from 37.7% in one year old stock to 73.8% in ewes that were older than six years, showing that acquired infections during the life of the animals is frequent and that environmental contamination by T. gondii oocysts must be significant. The median within-flock seroprevalence varied significantly across Scotland, with the lowest seroprevalence of 42.3% in the South and the highest seroprevalence of 69.2% in the far North of Scotland and the Scottish Islands, while the central part of Scotland had a seroprevalence of 57.7%. This distribution disequilibrium may be due to the spread and survival of oocysts on pasture and lambing areas. A questionnaire accompanying sampling of flocks identified farms that used Toxovax®, a commercial vaccine that protects sheep from abortion due to T. gondii infection. Only 24.7% of farmers used the vaccine and the vaccine did not significantly affect the within flock seroprevalence for T. gondii. The implications for food safety and human infection are discussed.