Astragalus is a crucial forage plant in the rangelands of northeastern Iran. However, identifying different species of Astragalus can be challenging due to their overlapping morphological features. In this study, we aimed to determine the relationships between 12 Astragalus species using both morphological and molecular traits. We also evaluated the effectiveness of DNA barcoding as a tool for identifying rangeland species of Astragalus in Northeast Iran. We examined five DNA barcodes, including three cpDNA regions (trnH-psbA, rpl32-trnL(UAG), and matK) and two nuclear sequences (ITS and ETS). We found that the plastid markers (rpl32-trnL(UAG, matK) were the most effective in differentiating between species. We also found that morphological data, as represented by the Neighbor-Net network and UPGMA dendrogram, had the potential to separate Astragalus species. We identified matK as the best and most accurate marker for barcoding, as trnH-psbA had some defects due to sequence size and alignment issues. Our study highlights the importance of using barcoding for quick and accurate recognition of plant species and shows the plastid markers were the most effective in differentiating between species.
