Objective To investigate genotypes, epidemiological characteristics, and possible  transmit mechanisms of resistance of PER type extended-spectrum β-lactamase in clinical Enterobacteriaceae isolates. Methods A total of 254 clinical isolates of Enterobacteriaceae with resistance to ceftazidime, cefotaxim, and cefepime were collected from June, 2009 to January, 2014 in Ruijin Hospital, Shanghai Jiao Tong University School of Medicine. The blaPER was identified by the PCR and sequencing. Conjugation experiments were performed for blaPER-positive strains and epidemiological analysis was conducted by the pulsed field gel electrophoresis (PFGE). The upstream and downstream sequences of PER gene were amplified by the PCR. Results A total of 14 clinical Enterobacteriaceae isolates carried the blaPER, including PER-1 type and PER-4 type. Two P. mirabilis isolates were successfully transferred by IncA/C conjugative plasmid. Nine isolates of blaPER-positive P. mirabilis showed 7 different PFGE patterns. The most common genetic environment of Enterobacteriaceae was ISCR1-blaPER-gst-abct and the genetic environment of 2 blaPER-positive P. mirabilis were ISPa12-blaPER-gst-like-ISPa13. Conclusion The blaPER was detected among a variety of clinical Enterobacteriaceae isolates. The upstream genetic environment of blaPER is given priority to the genetic environment with ISCR1 element, which may play an important role in the transmission of Chinese blaPER gene.