Theileriosis is parasitic infection causes by obligate intracellular protozoa of the genus Theileria. T. lestoquardi is the most virulent species in sheep and goats which causes a severe disease with a high morbidity and mortality rate. In this study the phylogenetic relationships between two local isolate of T. lestoquardi and nine T. lestoquardi global isolates as well as Babesia ovis out-group isolate were analyzed using the 18S rRNA gene sequence. The multiple sequence alignment analysis and neighbor joining phylogenetic tree analysis were performed by using ClustalW multiple sequence alignment online based analysis of 1098bp 18S rRNA gene was amplified by polymerase chain reaction. Phylogenetic analysis results of these gene sequences revealed that T. lestoquardi local isolates were closely related to T. lestoquardi Iran isolate (JQ917458.1) and two Iraq Kurdistan isolates (KC778786.1 and KC778785.1) more than other countries. This study represents the first report on the use of molecular phylogeny to classify T. lestoquardi obtained in Middle Region of Iraq.