This chapter discusses the use of DNA barcoding for biological identification, focusing on mitochondrial DNA sequences as markers for species differentiation. DNA barcoding involves analyzing short, standardized DNA segments to identify species, particularly in cases where traditional taxonomic methods are insufficient. The mitochondrial gene cytochrome c oxidase I (COI) has been widely used as a core barcode for animal species, with additional markers such as 16S rRNA and cytochrome b being used for complementary identification. The chapter highlights the importance of DNA barcoding in food safety, biodiversity monitoring, and forensic science, as well as its potential to replace or complement traditional taxonomic methods. The study presents methods for DNA extraction, PCR amplification, and sequencing of specific DNA fragments from various organisms, including Egyptian buffaloes, chickens, and predatory mites. The results show that DNA barcoding can effectively distinguish between closely related species, such as buffalo and cattle, and has potential applications in identifying unknown species. Phylogenetic analysis using DNA sequences has been used to construct evolutionary trees, revealing relationships between species and aiding in the identification of unknown organisms. The chapter also discusses the advantages of DNA barcoding, including its ability to provide standardized identification tools, reduce the burden on taxonomists, and facilitate biodiversity inventories. It highlights the importance of DNA barcoding in various fields, including biomedicine, agriculture, and environmental science. The chapter concludes with a discussion on the development of DNA barcoding in conjunction with genomics, emphasizing the need for standardized, short DNA sequences that can be easily generated and used for species identification. The study also explores the potential of using RNA data to infer DNA information, which could have implications for individual identity protection in research. Overall, the chapter underscores the significance of DNA barcoding in advancing species identification and biodiversity research.This chapter discusses the use of DNA barcoding for biological identification, focusing on mitochondrial DNA sequences as markers for species differentiation. DNA barcoding involves analyzing short, standardized DNA segments to identify species, particularly in cases where traditional taxonomic methods are insufficient. The mitochondrial gene cytochrome c oxidase I (COI) has been widely used as a core barcode for animal species, with additional markers such as 16S rRNA and cytochrome b being used for complementary identification. The chapter highlights the importance of DNA barcoding in food safety, biodiversity monitoring, and forensic science, as well as its potential to replace or complement traditional taxonomic methods. The study presents methods for DNA extraction, PCR amplification, and sequencing of specific DNA fragments from various organisms, including Egyptian buffaloes, chickens, and predatory mites. The results show that DNA barcoding can effectively distinguish between closely related species, such as buffalo and cattle, and has potential applications in identifying unknown species. Phylogenetic analysis using DNA sequences has been used to construct evolutionary trees, revealing relationships between species and aiding in the identification of unknown organisms. The chapter also discusses the advantages of DNA barcoding, including its ability to provide standardized identification tools, reduce the burden on taxonomists, and facilitate biodiversity inventories. It highlights the importance of DNA barcoding in various fields, including biomedicine, agriculture, and environmental science. The chapter concludes with a discussion on the development of DNA barcoding in conjunction with genomics, emphasizing the need for standardized, short DNA sequences that can be easily generated and used for species identification. The study also explores the potential of using RNA data to infer DNA information, which could have implications for individual identity protection in research. Overall, the chapter underscores the significance of DNA barcoding in advancing species identification and biodiversity research.