Background: Escherichia coli (EC), Escherichia fergusonii (EF), and Citrobacter freundii (CF) are clinically significant Gram-negative bacteria that are difficult to differentiate because of their shared 16S rRNA gene sequences. Methods: This study presents a novel approach utilizing adenylate kinase (adk) gene polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis for the precise discrimination of EC, EF, and CF. Analysis of adk sequences revealed unique restriction sites for BtsIMutI, BtgI, and AgeI restriction enzymes at nucleotide positions 93 and 96. Results: Theoretical predictions translated into distinct banding patterns during agarose gel electrophoresis following PCR/restriction digestion. Experimental validation with reference strains and clinical isolates, including 84 EC and CF strains, demonstrated the efficacy of this method for differentiating these species. Assay specificity was confirmed by selective digestion of adk amplicons with BtsIMutI for EC and AgeI for CF. Conclusion This molecular technique provides a rapid and accurate method to discriminate between closely related bacterial species and is promising for clinical diagnostics and epidemiological studies. Thus, our adk PCR/restriction digestion assay is a valuable tool for the advancement of bacterial typing methods for EC, EF, and CF, and contributes to the ongoing exploration of microbial diversity and epidemiology.