In poultry industry, mycoplasmas cause large economic losses. The pathogenic significance of mycoplasmas varies widely between species. In birds where vocalization is crucial for reproductive success, mycoplasmas seem to be absent. Birds with high mycoplasma prevalence like birds of prey and storks do not rely on their vocalization for reproduction. Corvids with an intermediate prevalence use other strategies besides vocalization for mating. It is hypothesized that mycoplasma prevalence and vocalization used for reproduction is evolutionary related. Barn swallows have two traits relevant for mating: tail feather length and vocalization. An intermediate prevalence is expected. Little is known about mycoplasmas in barn swallows and their role as vector or reservoir for poultry pathogenic mycoplasmas. This study investigates the prevalence of mycoplasmas in barn swallows and their relevance for mycoplasma transmission to poultry. Choanal swabs from 188 healthy barn swallow nestlings of 59 different nests from ten different colonies on farms were examined for mycoplasmas by cultivation and genus-specific PCR including molecular biological differentiation of the species. In total, in 31 of 188 barn swallows (16.49%) and in 14 of the 59 nests (23.73%), mycoplasmas were detected. The occurrence of mycoplasmas per colony ranged from 0 to 50% independent of poultry being kept on the farm. In all positive samples, Mycoplasma sturni was identified. Mycoplasma sturni seems not to be an obligatory pathogen for barn swallows and occurs with an expected intermediate prevalence in them, so the results support the described hypothesis and underline a minor role of barn swallows for mycoplasma infection in poultry.