Our study is based on the collection of blood and ticks from sheep in Iran and Italy. Polymerase chain reaction (PCR) testing was performed to target the 18S rRNA gene and RLB was performed using previously published probes. In Italy and Iran 78.7% and 76.0% of the sheep were PCR positive, which after sequencing and RLB showed that they were Theileria ovis and Theileria lestoquardi, respectively. Phylogenetic analysis was performed using the Clustal W multiple sequence alignment program and our sequences were compared with more than 50 others already published in the EMBL database. Our T. lestoquardi sequences linked with other T. lestoquardi sequences from Iran, Tanzania, and Sudan and Theileria annulata showed the importance of having species-specific probes between these two species. However, distinctive clades were found between T. lestoquardi ticks and those found in sheep blood. Italian T. ovis seemed to be closer to Theileria spp. from Namibia and Iran than with other T. ovis from Spain, Turkey, Tanzania, and Sudan adding some information to the controversy about this species. However, some confusion was found on the existing database where the location of pathogens, years, and species names was inaccurate and when available sequences were not always appropriately used. This article will discuss our results and some comparisons with other phylogenetic approaches.