AbstractBackground: Bacteria belonging to the genus Mycoplasma are small-sized, have no cell walls and small genomes. They commonly cause respiratory disorders in their animal hosts. Three species have been found in the respiratory tract of horses worldwide, that is., Mycoplasma (M.) equirhinis, M. pulmonis and M. felis, but their role in clinical cases remains unclear. Objectives: The aim of this study was to i) develop and validate tools to detect, isolate and identify different Mycoplasma spp. strains in clinical equine respiratory-tract specimens and ii) subsequently define the prevalence of the three species in France depending on sample types and horse characteristics (age, breed, sex). Study design: Validation of a workflow for mycoplasma diagnosis and subsequent prevalence study. Methods: Mycoplasma-free tracheal wash samples spiked with numerated strains and DNA dilutions were used to validate the culture methods and real-time PCR (rtPCR) assay. Isolated strains were identified by 16S rRNA gene sequencing. Prevalences were determined on a population of 616 horses with respiratory disorders, sampled in France in 2020. Results: In total, 104 horses (16.9%) were found to be positive for Mycoplasma spp. by at least one method. M. equirhinis was the predominant circulating species, accounting for 85% of the rt-PCR-positive samples and 98% of the 40 cultured strains. Main limitation: The proposed pre-enrichment procedure improves the sensitivity of detection but hinders the quantification of the initial mycoplasma load in the clinical specimens. Conclusions: Prevalence of mycoplasma varied with age, breed, and type of sample.