Mycoplasma gallisepticum (MG) is a major pathogen of the poultry industry throughout the world. MG causes chronic respiratory disease in chickens and infectious sinusitis in turkeys. Despite constant improvements in the biosecurity of the poultry industry in Iran, MG infection still occurs and causes significant economic issues. To evaluate genetic variability, 10 Iranian MG isolates along with 17 available sequences were characterized by gene-targeted sequencing (GTS) analysis of complete mgc2/pvpA genes. According to the findings, 21 different sequence types within the sample set of 27 strains were typed by this method. The discriminatory power of this typing assay was established to be 0.97. Although no insertions and deletions of nucleotides were observed in the mgc2 gene among the Iranian strains, different lengths of pvpA genes with 1086, 1095, and 1101 nucleotides were detected within direct repeats (DRs) 1 and 2. Generally, eight tetrapeptides Pro-Arg-Pro-Met/Gln/Asn were found in the DRs of PvpA. Analysis of the carboxyl ends of PvpA proteins exhibited various repeats of prolines. In the phylogenetic tree of partial and complete mgc2/pvpA genes, all Iranian MG isolates were clustered into two distinct groups. Because this typing assay could provide a higher discriminatory power than the previously reported GTS scheme of partial mgc2/ pvpA genes, these results can be considered a blueprint for future national control and diagnostic strategies. Furthermore, consistent surveillance with larger datasets will be needed to clarify the epidemiologic characteristics of MG outbreaks in different poultry hosts.