DNA barcoding is a method of species identification that revolutionized the way we study and understand biodiversity. With advancements in molecular genetics and DNA sequence archives, it has become possible to use short DNA sequences to identify species, even when the specimens are difficult to distinguish by traditional morphological methods. In this article, we explore the utility of DNA barcoding in the Tuensang ecosystem of Nagaland, India and evaluate its effectiveness for species identification, informing ongoing conservation of populations and species. A total of 62 species, which included insects, spiders, lizards, birds and mammals, were collected from Tuensang areas of northeast India and identified using DNA barcodes. DNA was extracted from muscle tissue and PCR was done with two pairs of primers targeting the mitochondrial COI gene. Sanger sequencing was employed and the obtained sequences were analysed to identify the species and reconstruct the evolutionary relationships amongst them. Our results provided molecular characterization of species from Tuensang areas of Nagaland for the first time.