Cytochrome c oxidase subunit I or COI in the mitochondrial genome (DNA Barcode) of goats was used to identify and differentiate two common breeds (beetal and berberi) and crossbreeds sampled in Punjab, Pakistan. This is the first study on the molecular taxonomy of the goat breeds of Pakistan. Sequencing of DNA barcode of the beetal goat showed a 99% similarity to Capra hircus isolate LS16 cytochrome c oxidase subunit 1 (CO1) gene. Beriberi goats showed a 99% similarity to Capra hircus breed Jining Qing goat mitochondrion. Identification of goat breeds via DNA barcoding may help in local genetic improvement and conservation programs, especially in pre-screening important breeds that can be considered for conservation in their pure form. However, more COI sequences should be determined from the native goat populations of Pakistan to improve reliability of using DNA barcodes to differentiate them from their exotic counterparts. Thus, the present study concluded that DNA barcoding can be used to confirm the species or breed origin of an unknown specimen and it is a reliable and practical tool to protect the local biodiversity of livestock genetic resources. Our results validated the effectiveness of barcoding for the identification of goat breeds.