Morphologically, 64 strongylid species have been described in equines. Co-infections are common, with up to 29 species reported in a single horse. Morphological identification of these species is time consuming and requires expert knowledge due to their similar appearance. Therefore, non-invasive identification methods are needed. DNA barcoding offers a rapid and reliable tool for species identification and the discovery of cryptic species for these most common parasitic nematodes of equines. In total, 269 cytochrome c oxidase subunit I (COI) gene and 312 internal transcribed spacer 2 (ITS-2) sequences from 27 equine Strongylidae species, including sequences from two uncharacterised species, Coronocyclus sagittatus and Triodontophorus tenuicollis, were generated and combined with COI and ITS-2 sequences data from six Cyathostominae species from previous studies. This study represents a comprehensive DNA barcoding analysis of 22 Cyathostominae and six Strongylinae species using mitochondrial COI gene and ITS-2 sequences. Maximum likelihood phylogenetic trees were constructed and the intra- and interspecific genetic distances for both markers were compared. Analysis revealed complex phylogenetic relationships. Para- and polyphyletic relationships were observed among most genera within Strongylinae and Cyathostominae. This challenges current morphological classifications. Although both markers showed overlapping pairwise identities in intra- and inter-species comparisons, COI had higher discriminatory power than ITS-2. Expanding the COI and ITS-2 reference database, including the first sequences for Coronocyclus sagittatus and Triodontophorus tenuicollis, improve a reliable species identification and advanced studies on Strongylinae and Cyathostominae diversity using barcoding and metabarcoding.
