Like humans, chickens can become sick from bacterial infections. Mycoplasma is a very small, slow-growing bacterium. There are multiple types of Mycoplasma that affect different animals, humans, and even plants and insects. In the current study, the whole genome of Mycoplasma synoviae was sequenced through the joint sequencing method of the third-generation PacBio and second-generation Illumina of the high-throughput sequencing platform, and the complete genome map of the Mycoplasma synoviae was successfully obtained. SMRT sequencing and assembly of whole genome: Canu v1.5/ WTDBG v2.2 software was used for preliminary assembly of filtered subreads. Pilon V1.22 software was used to further correct the assembled genome with high-quality second-generation data, and the genome with higher accuracy was finally obtained. Sequencing results showed that the Mycoplasma was composed of a 0.83Mb chromosome with a G+C percentage of 28.42%. Bioinformatics was used to predict the encoding protein genes, and it was found that Mycoplasma synoviae genome encoded a total of 1415 ORF open reading frames, including 1.73% of the repeats, 7 ribosomal RNAs, 34 transport RNAs, 4 non-coding RNAs and 12 pseudogenes. Mycoplasma synoviae genomes also contain 2 CRISPR, 1 gene island and 2 prophages. Using multiple strains of Mycoplasma gallisepticum, Mycoplasma iowae and Mycoplasma anatis as reference genomes, we constructed phylogenetic trees of 12 species of Mycoplasma. The genetic and taxonomic status of Mycoplasma synoviae strains were evaluated. The results showed that the sequencing strains of Mycoplasma synoviae and the model strain Mycoplasma synoviae WVU1853 were closely clustered together, suggesting the genetic relationship was relatively close and the sequencing strains were relatively conservative, and represent that they share the same genetic origin.