DNA barcoding uses specific DNA regions to identify species. For land plants, no standard barcode has been agreed upon. This study evaluated seven candidate plastid DNA regions (atpF-atpH, matK, rbcL, rpoB, rpoC1, psbK-psbI, and trnH-psbA) based on recoverability, sequence quality, and species discrimination. The recommended barcode is the combination of rbcL and matK. This two-locus barcode provides a universal framework for DNA-based species identification and helps discover overlooked plant species. The study used data from 907 samples across 550 species, including angiosperms, gymnosperms, and cryptogams. Universality was assessed by PCR and sequencing success, sequence quality by bidirectional sequencing and ambiguity, and discrimination by species differentiation. rbcL and matK showed high universality, good sequence quality, and strong species discrimination. While other loci had issues with sequencing success or sequence quality, rbcL and matK were the best overall. rbcL is well-characterized and easy to sequence, while matK has high discriminatory power but lower universality. The combination of rbcL and matK balances these factors, offering high universality, good sequence quality, and strong discrimination. This barcode is practical for large-scale sequencing and helps identify plant species, even from small or difficult-to-identify samples. It is also useful for biodiversity studies, conservation, and trade monitoring. Future improvements may include additional loci for better resolution, but the rbcL+matK barcode is recommended as a standard for land plants.DNA barcoding uses specific DNA regions to identify species. For land plants, no standard barcode has been agreed upon. This study evaluated seven candidate plastid DNA regions (atpF-atpH, matK, rbcL, rpoB, rpoC1, psbK-psbI, and trnH-psbA) based on recoverability, sequence quality, and species discrimination. The recommended barcode is the combination of rbcL and matK. This two-locus barcode provides a universal framework for DNA-based species identification and helps discover overlooked plant species. The study used data from 907 samples across 550 species, including angiosperms, gymnosperms, and cryptogams. Universality was assessed by PCR and sequencing success, sequence quality by bidirectional sequencing and ambiguity, and discrimination by species differentiation. rbcL and matK showed high universality, good sequence quality, and strong species discrimination. While other loci had issues with sequencing success or sequence quality, rbcL and matK were the best overall. rbcL is well-characterized and easy to sequence, while matK has high discriminatory power but lower universality. The combination of rbcL and matK balances these factors, offering high universality, good sequence quality, and strong discrimination. This barcode is practical for large-scale sequencing and helps identify plant species, even from small or difficult-to-identify samples. It is also useful for biodiversity studies, conservation, and trade monitoring. Future improvements may include additional loci for better resolution, but the rbcL+matK barcode is recommended as a standard for land plants.