BACKGROUND: Causing site direct mutation can be one of the efficient methods to evaluate the characteristics and properties of various genes. Brucellosis is the most common zoonotic infectious disease that would cause great economic losses. Thus, recognition of pathogenic and immunogenic factors in the genus Brucella can lead to control this health problem. Objectives: Considering the importance of site direct mutation in identification of genome structure and numerous ways to achieve this goal, Overlap Extension PCR is introduced as an improved technique for the removal and replacement of the gene target. Methods: For this study, with two-step PCR using specific primers, upstream and downstream fragments from target gene and antibiotic resistance cassette from plasmid pET28a (+), were reproduced and were connected to each other. The resulting fragment was cloned in specific position of pBluescriptIISK(-) plasmid by the restriction enzymes. Then, the construction was transferred into the genome of Brucella abortus by electroporation method. Results: Fusion PCR product was obtained without any change in the nucleotide sequence and then it was cloned into pBluescriptIISK (-) plasmid, finally the construction was replaced and the target gene was deleted. Conclusions: The results of this study show that the Overlap Extension PCR is an optimized and modified technique to create mutations in the bacterial genome structure and can easily be used in the family Brucella.