Contemporary molecular biology provides information about entire genome sequences, which are used in fields such as medicine, pharmacogenetics, and nutrigenomics. Apart from the DNA located in cell nuclei, studies of the DNA in cell organelles, such as chloroplasts and mitochondria, are important as well. Analysis of selected mtDNA fragments has become the basis for molecular barcoding – species identification based on polymorphisms in the nucleotide sequence. Of particular importance in identification analyses are fragments encoding genes of the respiratory chain – cytochrome b and cytochrome oxidase, with intraspecific similarity but at the same time high interspecific variation, owing to which mtDNA analysis has found an application in many fields of science, such as forensic genetics and molecular anthropology. An unquestionable advantage of molecular methods over traditional ones is that identification can be based on trace quantities of material, including highly processed or degraded material (bone fragments, teeth, or fur). Moreover, bioinformatic tools supporting molecular analyses limit the need for testing of reference material, as the results can be compared to sequences deposited in databases. Given the increasing availability of molecular methods and the decrease in costs resulting from the development of the technology, species identification based on polymorphism in mtDNA may become a routine research method in the fields of ecology, molecular archaeology, molecular taxonomy, and forensics. This work is a review of areas in which mitochondrial DNA is used for species and individual identification. The analysis methodology and examples of the practical applications of mtDNA studies are discussed as well.