The aim of this study was to investigate the presence of antimicrobial resistance in E. coli strains isolated from red deer (Cervus elaphus) and to determine their phylogroup distributions. In the study, 123 E. coli were isolated from deer feces. The highest rate of resistance was determined against sulfonamide (n = 72, 58.54%), streptomycin (n = 54, 43.90%), ampicillin (n = 33, 26.83%), and tetracycline (n = 14, 11.38%) and multidrug resistance was detected in 20 isolates (16.26%). None of the isolates were phenotypically resistant to fluoroquinolones and imipenem. Antibiotic resistance genes of the isolates phenotypically found to be intermediate-susceptible and resistant to sulfonamide, tetracycline, and streptomycin were analyzed by PCR. As a result of genotypic evaluation, tetA, aadA, and strA-strB genes were detected in three different isolates, and the tetB gene was found in seven isolates. The other tested genes, i.e. tetC, sul1, sul2, and sul3, were not detected. The phylotyping revealed that 30.89% of the isolates belonged to phylogroup D, followed by phylogroup B1 (28.46%), A (9.76%), B2 (7.32%), E (6.50%), Clade I (6.50%), and phylogroup F (3.25%). In this study, the presence of antibiotic resistance and phylogenetic classification were reported for the first time in E. coli isolated from deer living in isolated areas without contact with humans and antimicrobial treatment in Türkiye. The antimicrobial resistance rates and the presence of some resistance genes in deer show that wildlife also plays an important role in public health.