Specific oligonucleotide probes were designed to develop a new and highly sensitive reverse line blot assay to detect and identify simultaneously different Theileria and Babesia species in horses. The amplified hypervariable V4 region of the 18S rRNA gene was hybridised against different generic and species-specific probes. The survey was conducted over 243 samples of equine blood divided into three different groups: group 1, 24 horses presented as possible clinical piroplasmosis; group 2, 181 clinically healthy free-ranging horses exposed to ticks; group 3, 38 riding horses with unrelated pathologies and low or no contact with ticks. The study demonstrated a high piroplasm prevalence in the first two groups of animals. Two Theileria genotypes sharing 96.8% similarity between their 18S rRNA gene sequences and two Babesia genotypes sharing 97.4% similarity, were identified. The biologic meaning of such genotypes is discussed in terms of their phylogenetic relationships and potential pathogenicity.