Temperature-sensitive strains of Mycoplasma gallisepticum, M. synoviae and M. hyopneumoniae, created using chemical mutagenesis, have proven to be effective vaccines against the three major mycoplasmoses causing disease in poultry and pigs. The use of these vaccines in poultry has significantly reduced reliance on antimicrobial therapy, with a consequent reduction in usage of macrolides. Recent advances in development of methods for genetic manipulation of mycoplasmas has enhanced our capacity to identify virulence genes, offering the prospect of the development of novel, rationally attenuated vaccines in the future.