[Objective] The purpose of this research was to analyze the biological characteristics of isolates NM1108-1 and NM1108-5 obtained from gadfly in China, and to determine the taxonomy of the two isolates. It provided more information about interaction between spiro-plasma and their host. [Methods] The gadfly spiroplasmas were isolated and cultured by con-ventional methods. Dark field and transmission electron microscopy were used to examine the morphology and movement of spiroplasma isolates. The taxonomy of the two isolates was evaluated according to their biological and phylogenetic characteristics. [Results] A large number of spiroplasmas were isolated from gadfly, and two representative strains NM1108-1, NM1108-5 exhibited helical form in logarithmic phase. Both the spiroplasmas from gadfly could use glucose, sucrose and D-fructose as a carbon and resisted to penicillin, but did not hydrolyse urea or arginine. The phylogenetic analysis based on 16S rDNA and ITS sequences showed that both isolates NM1108-1 and NM1108-5 belonged to the Apis clade but were di-vided into two different branches. NM1108-1, NM1108-5 were clustered into S. turonicom, S. gladiatoris respectively. [Conclusion] The isolates of NM1108-1 and NM1108-5 were initially determined as S. turonicom and S. gladiatoris respectively according to their biological and phylogenetic characteristics. This was the first report about the spiroplasmas from gadfly in China.