Mycoplasma synoviae expressed variable lipoprotein haemagglutinin (VlhA) is believed to play a major role in pathogenesis of the disease by mediating adherence and immune evasion. The aim of this study was sequencing Iranian M. synoviae isolates for the detection of nucleotide variation in the M. synoviae vlhA gene. Using oligonucleotide primers complementary to the single-copy conserved 5´ end of vlhA gene, amplicons of ~400 bp were generated from 10 M. synoviae isolated from commercial broiler chicken farms in Iran, afterward the conserved domain of the vlhA gene of M. synoviae was sequenced and analyzed for Iranian isolates. The results showed that, there was a complete concordance between all Iranian isolates nucleotide sequence (1-386 nt). In comparison with vaccine MS-H strain sequence, all Iranian isolates; entire vlhA sequence downstream of nucleotide 386 was different. It was also observed in all Iranian M. synoviae isolates, point mutations and frame-shift mutation. This study was demonstrated a difference between Iranian isolates and live commercial vaccine MS-H strain. Furthermore, these data indicated that changes in the vlhA gene sequence could introduce into the expressed vlhA gene amino acid codons and effective in pathogenesis rate in flocks.