Mycoplasma synoviae (MS) is an important pathogen of chickens and turkeys. In recent years sequence analysis of the partial MS variable lipoprotein and hemagglutinin A (vlhA) gene PCR product has been utilized routinely for MS strain genotyping. Several PCR assays have been proposed for the amplification of the conserved upstream region of the MS vlhA gene; however, in several clinical instances the published assays failed to generate vlhA PCR products from confirmed MS-positive cases. These occurrences hindered our capability to genotype those cases. In silico analysis of the published MS vlhA PCRs raised concerns, which were addressed by the design of revised MS vlhA PCRs. The published and revised assays were tested for their relative sensitivity and specificity with laboratory and clinical MS-positive samples. One of the revised MS vlhA PCRs (revised Hong) was demonstrated to be more sensitive and specific, and amplified all clinical samples analyzed in this study.