Canine infectious respiratory disease (CIRD) is a complex infection that occurs worldwide predominantly in kennelled dogs, and several bacterial and viral micro-organisms have been associated with outbreaks of CIRD. However, few studies have comprehensively examined the species of mycoplasma present in healthy dogs and those with CIRD. As part of an extensive study investigating the micro-organisms involved in CIRD, the species of mycoplasma present throughout the respiratory tract of dogs with and without CIRD were determined. Mycoplasmas were cultured from tonsillar, tracheal and bronchial lavage samples, and identified to the species level by PCR and sequencing. Mycoplasma cynos was demonstrated on the ciliated tracheal epithelium by in situ hybridization and was the only mollicute found to be associated with CIRD, but only in the lower respiratory tract. Isolation of M. cynos was correlated with an increased severity of CIRD, younger age and a longer time in the kennel.