Following Clinical and Laboratory Standards Institute (CLSI) recommendations, the susceptibilities of twelve traditional or new recommended antimicrobial agents were evaluated by the agar microdilution method in 1012 strains of Yersinia pestis isolated from 1943 to 2017 in 12 natural plague foci in China. A clinical Y.pestis isolate (S19960127) isolated from a pneumonic plague outbreak in 1996 in Tibet Autonomous Region exhibited high-level resistance to streptomycin (the MIC was 4,096 mg/L). The strain S19960127 was still sensitive to other eleven antibiotics, i.e. ciprofloxacin, ofloxacin, kanamycin, chloramphenicol, ampicillin, ceftriaxone, cefuroxime, trimethoprim-sulfamethoxazole, tetracycline, spectinomycin and moxifloxacin. The remaining 1011 Y.pestis strains in this study demonstrated susceptibility to above mentioned twelve antimicrobial agents. Streptomycin is used as the first-line antibiotic against plague in many countries include China, thus antimicrobial sensitivity surveillance of Y.pestis isolates, including dynamically monitoring streptomycin resistant during various clinical plague treatment should be carried out routinely.