Abstract:[Background] Acinetobacter spp. are common opportunistic pathogens of hospital infection, and the emergence of drug-resistant strains of Acinetobacter poses a serious threat to public health. [Objective] To understand the prevalence and drug resistance of Acinetobacter spp. in the pet hospital environments in Shanghai. [Methods] Environmental samples were collected from pet hospitals in Shanghai, and the isolated strains were identified by a mass spectrometer for bacterial identification. The minimum inhibitory concentration (MIC) was determined by the broth dilution method, and then the genome sequence and genetic characteristics of a carbapenem-resistant isolate were analyzed. [Results] A total of 124 strains of Gram-negative bacteria were isolated from 730 environmental samples, and 14 strains were identified as Acinetobacter spp., of which 9 strains were A. lwoffii. All the 14 Acinetobacter strains were sensitive to minocycline. Two A. pittii strains and two A. lwoffii strains demonstrated multidrug resistance to carbapenems. Five A. lwoffii strains were resistant to sulfamethoxazole trimethoprim, and one A. johnsonii strain was resistant to ciprofloxacin. The results of genome sequencing showed that the A. pittii strain with multidrug resistance to carbapenems carried drug resistance genes such as NDM-1, OXA-500, and OXA-727. The multilocus sequence typing (MLST) results showed that the isolate was of ST1037 and the single nucleotide polymorphism (SNP) analysis revealed that the isolate was close to A. pittii AP43 isolated from clinical urine in Zhejiang. [Conclusion] The drug resistance rate of Acinetobacter spp. in the environments of five pet hospitals in Shanghai was <40%, and there was multi-drug resistance (Five Acinetobacter strains were resistant to ≥3 drugs). A. pittii carried multiple drug resistance genes.