Bovine theileriosis is a tick-borne disease caused by one or more hemoprotozoan parasites of the genus Theileria. In the past, Theileria infection in cattle in Australia was largely asymptomatic and recognized to be associated with Theileria buffeli. However, outbreaks of theileriosis have occurred in beef and dairy cattle in subtropical climatic regions (New South Wales) of Australia. There is also one published report of a recent theileriosis outbreak in a beef farm near Seymour in the southeastern state of Victoria. In order to gain an improved insight into the genetic composition of Theileria populations following this outbreak, we undertook herein an integrated PCR-coupled mutation scanning-sequencing-phylogenetic analysis of sequence variation in part of the major piroplasm surface protein (MPSP) gene within and among samples from cattle involved in the outbreak. Theileria DNA was detected in 89.4% of 94 cattle in the Seymour farm; the genetic analysis showed that the ikeda and chitose genotypes representing the Theileria orientalis complex were detected in 75 and 4.8% of 84 infected cattle, respectively, and that mixed populations of these two genotypes were found in 20.2% of infected cattle. Given unpublished reports of a significant increase in the number of outbreaks in Victoria, future investigations should focus sharply on elucidating the epidemiology of Theileria to subvert the economic impact on the cattle industry in this state. Although used here to explore genetic variation within the T. orientalis complex in Australia, a mutation scanning-based approach has broad applicability to other species of Theileria in other countries.