BackgroundAvian mycoplasmas are known pathogens, which cause severe economic losses in poultry flocks. PCR is a rapid, sensitive, and less expensive diagnostic tool than culture for the identification of mycoplasmas in poultry farms. The objective of this study was to determine by PCR the presence of Mycoplasma spp., Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and Mycoplasma pullorum (MP) in laying hens located in the Sfax region, in the South of Tunisia.ResultsA total of 781 tracheal swabs were collected from 13 laying-hen farms without clinical signs at the date of sampling. MP was detected by a newly described specific PCR assay. The prevalence calculated from PCR results at the flock level was 100% for Mycoplasma spp., 0% for MG, 84.6% for MS and 61.5% for MP. The overall prevalence at the animal level was 38.7% for Mycoplasma spp., 0% for MG, 25% for MS and 6.4% for MP. The overall prevalence of 100% of avian mycoplasmas in laying-hen farms (38.7% prevalence at the animal level) shows an alarming situation.ConclusionsThese results underline the importance of monitoring the emergence and spread of Mycoplasma strains in farms in order to decrease economic losses due to mycoplasmoses.