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Objective  Grasslands are widespread ecosystems that fulfil many functions. Plant species richness (PSR) is known to have beneficial effects on such functions and monitoring PSR is crucial for tracking the effects of land use and agricultural management on these ecosystems. Unfortunately, traditional morphology-based methods are labor-intensive and cannot be adapted for high-throughput assessments. DNA barcoding could aid increasing the throughput of PSR assessments in grasslands. In this proof-of-concept work, we aimed at determining which of three plant DNA barcodes ( rbcLa ,  matK  and  trnH - psbA ) best discriminates 16 key grass and legume species common in temperate sub-alpine grasslands.    Results  Barcode  trnH - psbA  had a 100% correct assignment rate (CAR) in the five analyzed legumes, followed by  rbcLa  (93.3%) and  matK  (55.6%). Barcode  trnH - psbA  had a 100% CAR in the grasses  Cynosurus cristatus ,  Dactylis glomerata  and  Trisetum flavescens . However, the closely related  Festuca, Lolium  and  Poa  species were not always correctly identified, which led to an overall CAR in grasses of 66.7%, 50.0% and 46.4% for  trnH - psbA ,  matK  and  rbcLa , respectively. Barcode  trnH - psbA  is thus the most promising candidate for PSR assessments in permanent grasslands and could greatly support plant biodiversity monitoring on a larger scale.

DNA barcode trnH-psbA is a promising candidate for efficient identification of forage legumes and grasses