Strawberry (Fragaria x ananassa Duch.) is one of the high-economic soft fruit crops, which plays an important role in farmers' incomes in Turkey. In a disease survey conducted in 2018 and 2019 growing seasons, typical disease symptoms resembling fire blight caused by Erwinia amylovora were observed in Yozgat and Mersin province of Turkey. The diseased strawberry plants showed intense black to brown necrotic areas in the midribs and veins on the leaf area and the attached stems. Twelve strains of E. amylovora were isolated from naturally infected strawberry plants on the basis of disease symptoms. The strains were initially characterized based on physiological, biochemical characters, and pathogenicity tests and identified using MALDI TOF MS analyses. All strains were identified as E. amylovora based on their protein fingerprint patterns obtained by the MALDI TOF MS system. The identification of representative strains was further confirmed by PCR amplification of a specific region by using taxon-specific primers, sequencing 16S rDNA and 3 housekeeping genes (gapA, mdh and recA). Obtained partial nucleotide sequences of each gene were submitted to BLAST analysis and strains shared 100% (for gapA), 99.45% (for mdh) and 100% (for recA) nucleotide identity with each other and sequence of complete genomes of E. amylovora strains available in GenBank. Constructed phylogenetic trees with concatenated sequences of gapA, mdh and recA genes allowed distinctly to separate strawberry E. amylovora strains from other Erwinia species. Representative bacterial strains were further tested for copper and streptomycin sensitivity. None of the strains grown on the nutrient medium containing 2.56 mM copper sulphate and 5 ppm streptomycin sulphate indicated that strawberry isolates are sensitive to copper and streptomycin for now. This study is the first report of a natural outbreak of bacterial fire blight disease caused by E. amylovora on strawberries in Turkey. Since disease is transmitted by bees, the presence of the pathogen may seriously influence local strawberry production in the future.