Objective To establish a novel PCR method that can differentiate the two biotype of Mycoplasma urealytium rapidly based on the disparities of parC gene sequences for clinical routine examination.Methods Design two pairs of specific primers and probes for the targeted gene according to the differences of the parC gene sequences from 14 standard serotypes.The specificity of this amplification was verified by detecting two Mycoplasma urealyticum standard strains including serovar1 and serovar4,50 clinical isolated strains ( 12 are Uu strains and 38 are Up strains) and 7 common bacterias from vagina.Collected 70 swab specimens from patients of Sexually Transmit Department with urogenital inflammation symptom and 71 swab specimens from the gynecology health examination population.All those specimens were detected by culture and our method respectively.The sensitivity of this method was evaluated by comparing with the culture.Differences of the infection rate and distribution of biotypes between different populations were analyzed using statistical software.Results Standard strains of Mycoplasma urealyticum and clinical isolates can be typed into two species by the PCR method without nonspecific amplification.The sensitivity of PCR method is much higher than that of culture ( P＜0.05).The infection rates of Uu,Up and the mixing were 8.57％,61.40％,24.30％ respectively for the patients with vaginitis.However,it was 8.80％,67.60％,8.45％ respectively for the gynecology health examination population.There is a significant difference of the total infection rate between the two population(P＜0.05).It showed no significant difference with the distribution of the two types of simple infection in the two groups ( P＞0.05).But the rate of mixing infection is much higher in the patients with vaginitis ( P＜0.05 ).Conclusion The pathology of Mycoplasma urealyticum may be related to the mixed infection of different biotypes.