Abstract: OBJECTIVE To investigate the mycoplasma infection and drug resistance of patients with non gonococcal urethritis in hospital, so as to provide references for clinical treatment. METHODS A total of 152 cases of non gonococcal urethritis patients in a hospital from Jan. 2013 to Dec.2015 were selected as research objects. The mycoplasma culture and drug susceptibility testing were carried out, and changes of the drug resistance of mycoplasma to various types of antimicrobials were analyzed. RESULTS The positive rate of mycoplasma culture increased year by year from 2013 to 2015, but the proportion of all kinds of mycoplasma did not change significantly, most of them were Uu, followed by Uu + Mh, and Mh was the least. From 2013 to 2015, different sources of mycoplasma from high to low were cervical secretions and male urethral secretions. Mycoplasma infection rate was significantly higher in women than men, and was the highest among those aged 21-30 years old, followed by 31-40 years old, and the difference was significant (P<0.05). From 2013 to 2015, there was no significant difference in the resistant rates of Uu+Mh to doxycycline, minocycline, doxycycline, and josamycin, and Uu to ciprofloxacin. The resistant rates of Uu+Mh and Uu to doxorubicin, erythromycin cydocarbonate, azithromycin, clarithromycin, roxithromycin, erythromycin, levofloxacin, sparfloxacin, and thiamphenicol increased year by year, and the difference was significant (P<0.05). CONCLUSION Mycoplasma infection is more common in women aged 21-30 years, the incidence is increasing year by year, and the resistance to the various types of commonly used antimicrobial drugs is increasing year by year. Doxycycline, minocycline, and josamycin become the first choice in clinic, and it can select appropriate antibiotics for treatment based on mycoplasma culture and drug susceptibility.