<p>Fosfomycin is a well-known antibiotic that exhibits broad-spectrum activity against various bacterial pathogens, including gram-negative strains and some gram-positive strains such as staphylococci. The use of parenteral fosfomycin has been recently revised because the antibiotic has been found to effectively manage serious infections caused by multidrug-resistant pathogens. The occurrence of fosfomycin resistance could threaten the reintroduction of this antibiotic for the treatment of bacterial infections. In this study, a total of 24 fosfomycin-resistant <i>Escherichia coli</i> isolates obtained from urine samples were used to investigate the prevalence and molecular epidemiology of plasmid-mediated fosfomycin resistance genes. The replication origins of the conjugative and transformant plasmids obtained from the isolates were examined using the replication origin determination method based on the polymerase chain reaction (PCR). Through the PCR process performed with the <i>fosA</i>, <i>fosA3</i>, <i>fosB</i>, <i>fosC</i>, <i>fosC2</i>, and <i>fosX</i> genes to determine fosfomycin resistance, one out of 24 samples was found to be <i>fosA3</i> gene-positive. A Class-1 integron gene was detected in three fosfomycin-resistant <i>E. coli</i> isolates, while no Class-2 integrons were detected in any isolate. The conjugation experiments demonstrated that the <i>fosA3</i> gene was transferable in one isolate that also carried the <i>blaTEM</i>, <i>blaCTX-M-15</i>, and <i>aac(6′)-ib-cr</i> genes. Through plasmid isolation in the transconjugant <i>E. coli</i> isolates, it was determined that the <i>E. coli</i> isolate FF21 carried fosfomycin resistance on the plasmid. To ensure the continued effective use of fosfomycin as a treatment option, fosfomycin resistance needs to be detected and closely monitored. Given the global rise in plasmid-transmissible genes, we anticipate a growing resistance to fosfomycin in the near future.</p>