Equine piroplasmosis is caused by two haemoprotozoan parasites: Babesia caballi and Theileria equi. Negative economic impact on international trade has been associated to endemic sites. This is the reason why carrier detection requires reliable diagnostic methods. Various diagnostic modalities can be used alone or in combination including PCR. However, genetic variation of commonly used genes is still of debate. The aim of this research was to sequence the β-tubulin gene of a B. caballi strain from Spain and to compare it with known β-tubulin sequences.DNA was isolated from a cryopreserved strain from Spain and acute and chronic carrier horses. Firstly, degenerated primer pairs were designed based on GenBank sequences of different Babesia and Theileria species for sequencing. The primers were redesigned to amplify both parasites, simultaneously. Finally, a species-specific primer pair for B. caballi was designed and a Restriction Fragment Length Polymorphism-PCR (PCR-RFLP) assay performed to know the difference of known B. caballi strains.We provided new insights of the β-tubulin gene and a good molecular coverage of this gene, contributing with a number of useful primers to amplify T. equi and B. caballi. Moreover, PCR-RFLP assays based on the exon II of this gene confirmed the causative B. caballi strain in Spanish horses.We reported useful primer pairs for diagnostic and a new sequence of the β-tubulin gene of B. caballi, which will facilitate the development of future assays and the detection of infected horses, preventing thus the spread of this disease worldwide.