Mycoplasma synoviae (MS), a remarkable pathogen in poultry, causes subclinical infection of the upper respiratory tract and an infectious synovitis, especially in the tendon sheaths and synovial membranes of joints. Because the specific detection of MS 16S rRNA gene–based PCR was unsuitable for strain differentiation, vlhA gene–based PCR was designed to differentiate the MS strains. The vlhA gene of MS encodes for hemagglutinin and other immunodominant membrane proteins involved in colonization, antigenic variations, and virulence. Sequence analysis of the vlhA gene based on the nucleotide insertion/deletion of the proline-rich repeat (PRR) region and the nucleotide polymorphisms of the RIII region in vlhA gene fragments was useful for typing and subtyping of MS strains. This study aimed to characterize the Thai MS field isolates and to differentiate the field and vaccine strains in Thailand by using sequence analysis of the partial vlhA gene. In total, 20 MS field isolates submitted from registered chicken farms in Thailand during 2015 were identified as Type C1 (n = 1), C2 (n = 4), E1 (n = 9), E2 (n = 1), and L (n = 5). The results revealed that six of the nine isolates resulting in respiratory signs were Type E1. In addition, four isolates from lame chickens showing joint swelling were identified as Type L, with a length of 105 nucleotides. This study provides the first molecular data of Thai MS isolates and the first evidence of Type L for being an arthropathic strain that differs from a previous study demonstrating that only MS Type B, with a longer PRR of 135 nucleotides, could be highly invasive strains and associated with infectious synovitis in chickens. Furthermore, one farm showed coinfection of MS Types E and L, but most of the farms were affected by only one type of MS. The results indicated that sequence analysis of the partial vlhA gene can be used as a tool for tracing MS characterization.