The aim was to determine the prevalence and antibiotics resistance of Uroaplasma urealyticum (U.urealyticum) and Mycoplasma hominis (M. hominis) isolated in a southwestern area of china. A total of 1093 patients with Chronic Prostatitis/ Chronic Pelvic Pain Syndrome (CP/CPPS) were included and Expressed Prostatic Secretion (EPS) were collected from the subjects. Antibiotic resistance tests were conducted by using the mycoplasma kits. Of the individuals studied, 17.29% (189/1093) and 3.66% (40/1093) samples were respectively identified to be positive for U.urealyticum and M. hominis in EPS. U.urealyticum were less than 10% (0- 8.99%) resistance to doxycycline, minocycline, azithromycin and josamycin, while they were higher than 60% (60.85%-73.54%) resistance to ciprofloxacin, ofloxacin and gatifloxacin, and the resistance incidence of erythromycin to U.urealyticum was 39.15%). The resistance rate of M. hominis was less than 10% (0-2.5%) to doxycycline, minocycline and josamycin, while they were higher than 60% (62.50%- 87.50%) to azithromycin, erythromycin, ciprofloxacin and ofloxacin, and that of gatifloxiacin was 32.50%. In conclusion, testing for both U.urealyticum and M. hominis in EPS of patients with CP/CPPS should been encouraged and doxycycline, minocycline and josamycin were recommended to treat infections of these strains in patients with CP/CPPS in a southwestern area of china. Further investigations should be focus on the new promising antibiotics against M. genitalium due to increasing resistance of antimicrobial.