DNA barcoding is a very useful and promising approach to the identification of biological species in which DNA sequences from a tissue sample of the target organism serve as the basic key for information retrieval. Old existing software for evolutionary analysis of DNA (specific or genomic) sequences was developed for taxonomic and phylogenetic analysis. Those algorithms do not deliver appropriate analysis for the rapid but accurate analysis needed for DNA barcoding. In this chapter, strength and limitation of DNA barcoding, laboratory methodologies of DNA barcoding, bioinformatics tools, and databases used for DNA barcoding have been discussed. The basic process of DNA barcoding follows two main steps: (1) building the DNA barcode library (specific regions or whole genomic) of known organisms and (2) matching those barcode sequences of the unknown target samples against the barcode library for species identification. Although DNA barcoding as a technology has been in use for less than a decennary, it has grown rapidly in terms of the number of DNA sequences produced as barcodes as well as its applications. This chapter provides the latest facts on generating, analyzing, and applying DNA barcodes across the evolutionary tree of life from animals and fungi to algae and plants and diagnosis of the biological diversity.