Spirlins of the genus Alburnoides are widespread fishes, which taxonomy has been rapidly developing in recent years. Mitochondrial cytochrome c oxidase subunit I (COI) was used as DNA barcode marker to create a reference dataset of Caucasian Alburnoides and to test its barcoding efficiency. All four previously known Caucasian species of Alburnoides were confirmed as valid species with high genetic distances to sister species as well confirmed as Caucasian endemics. Alburnoides samiii, previously known from Sefidroud basin (Iran), was discovered in Transcaucasia. The accuracy of species identification of Ponto-Caspian Alburnoides by DNA barcodes was 100%. In addition, one potentially new species within A. gmelini was revealed. Despite the limited ability of COI to infer phylogenetic relationships, study provided evidence that Ponto-Caspian lineage of Alburnoides includes significantly larger number of species from Caspian Sea basin and inland basins of Central Asia.