The emergence and dissemination of resistance to clinically important antimicrobials in wild animals is of great concern. The aim of our study was to reveal the prevalence and intraspecies dissemination of quinolone-resistant Escherichia coli (QREC) in sika deer (Cervus nippon) in Nara Park, a famous tourist spot in Japan. Fecal samples were collected from 59 wild deer in Nara Park between July and October 2018. We isolated QREC using deoxycholate-hydrogen sulfide-lactose agar containing nalidixic acid and subjected it to antimicrobial susceptibility testing. The mutations in the quinolone resistance-determining region (QRDR) of the gyrA and parC genes of the isolates were analyzed and fragment patterns of genomic DNA were compared by pulsed-field gel electrophoresis (PFGE). A total of 105 QREC isolates were obtained from 41 deer (70%). All isolates had mutations within the QRDR. Other than quinolone resistance, QREC isolates also showed resistance to various other antimicrobial agents. The QREC isolates were classified into 15 PFGE clusters, of which seven were observed in multiple deer. Our results suggest clonal transmission of QREC in a high-density deer population. Spread of QREC in deer inhabiting a tourist location could have potential impact on public health.