We induced sulfadiazine resistance in two sulfadiazine sensitive strains of Toxoplasma gondii, RH (Type I) and ME-49 (Type II) in vitro by using drug pressure. At first, sulfadiazine susceptibility of the two sensitive strains and two naturally resistant strains of T. gondii was evaluated on Vero cells using an enzyme-linked immunosorbent assay (ELISA). The IC 50 values of sulfadiazine were 77μg/mL for RH, 51μg/mL for ME-49 and higher than 1000μg/mL for the two natural resistant strains. Secondly, induced resistance of the strains by gradually increase sulfadiazine concentration was verified by this test, which resulted IC 50 values at higher than 1000μg/mL. In conclusion we developed in vitro two sulfadiazine resistant strains called RH-R SDZ and ME-49-R SDZ . These strains resistant to sulfadiazine would be useful to characterize resistance mechanisms to sulfadiazine.