Four avian mycoplasmas are commonly recognized as poultry pathogens: Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Mycoplasma meleagridis (MM), and Mycoplasma iowae (MI). The avian mycoplasmas are associated with respiratory disease, synovitis and arthritis, poor performance, skeletal deformities, and embryo mortality. Three main approaches are used for the diagnosis of avian mycoplasmosis: isolation and identification, detection of antibodies, and molecular detection of the organism's nucleic acid by PCR. In recent years real-time PCR technology has revolutionized the way clinical microbiology laboratories diagnose infectious diseases, but so far only a limited number of diagnostic real-time PCRs have been proposed for avian mycoplasma diagnostics. We developed a complete set of reliable diagnostic real-time TaqMan PCR assays for the four pathogenic avian mycoplasmas. The selected genomic targets of the developed assays were species specific and intraspecifically conserved and included the 16S–23S intergenic spacer Region of MS and MM, the upstream Region to the 16S ribosomal DNA of MI, and highly conserved foci of the mgc2 gene of MG. The four assays were demonstrated to be highly specific and sensitive to their target avian mycoplasma, with detection limits of one copy per reaction mix for the MG assay and 10 copies per reaction mix for the MS, MM, and MI assays. We believe that the incorporation of the developed assays in avian mycoplasma diagnostics will contribute to the accuracy, efficiency, and feasibility of diagnosis of these pathogens.