Mycoplasma gallisepticum (MG) is an economically important pathogen of poultry worldwide, causing chronic respiratory disease in chickens and turkeys. Differentiation of MG strains is critical, especially in countries where poultry flocks are vaccinated with live vaccines. In this study, oligonucleotide primers were designed based on a region preceding the trinucleotide repeat of a member of the vlhA gene family, and amplicons of 145-352 bp were generated from cultures of 10 different MG strains, including the ts-11, F and 6/85 vaccine strains. High-resolution melting (HRM) curve analysis of the resultant amplicons could differentiate all MG strains. Analysis of the nucleotide sequences of the amplicons from each strain revealed that each melting curve profile related to a unique DNA sequence. The HRM curve profiles (for ts-11) remained consistent after at least five passages under laboratory conditions. PCR-HRM curve analysis of 33 DNA extracts derived from respiratory swabs, or mycoplasma cultures grown from respiratory swabs, of ts-11 vaccinated commercial or specific pathogen-free chickens identified all these specimens, according to their sequences, as ts-11. The potential of the PCR-HRM curve analysis was also shown in the genotyping of 30 additional MG isolates from Europe, the USA and Israel. The results presented in this study indicate that PCR followed by HRM curve analysis provides a rapid and robust technique for genotyping of MG isolates/strains using both MG cultures and clinical swabs.