This study investigates the effectiveness of DNA barcoding for identifying plant species in two biodiversity hotspots: Mesoamerica and southern Africa. The researchers collected over 1,600 plant samples and tested eight potential DNA barcodes to determine which is most suitable for species identification. They identified a portion of the plastid *matK* gene as a universal DNA barcode for flowering plants due to its adequate rate of variation, ease of amplification, and alignment. They also demonstrated the applicability of DNA barcoding for biodiversity inventories, particularly in identifying cryptic species and those listed in the Convention on International Trade in Endangered Species (CITES). The study found that while the *trnH-psbA* region also performs well, its molecular evolution is complex, making it less reliable than *matK*. The *matK* gene was found to be the most suitable barcode, as it exhibits a "barcoding gap" between intra- and interspecific variations, allowing for clear species discrimination. The researchers also tested the utility of *matK* in identifying cryptic species, such as the orchid *Lycaste cf. tricolor*, which was found to be distinct from the typical *L. tricolor* based on morphological and genetic differences. The study highlights the importance of DNA barcoding in biodiversity conservation, particularly in regions with high species diversity and taxonomic challenges. It also underscores the need for further research to identify complementary barcodes and to improve the identification of difficult lineages. The results suggest that DNA barcoding with *matK* alone or in combination with *trnH-psbA* can achieve over 90% correct species identification, making it a valuable tool for biodiversity inventories and conservation efforts. The study concludes that *matK* is a preferred universal DNA barcode for flowering plants, with *trnH-psbA* serving as an alternative or complementary barcode.This study investigates the effectiveness of DNA barcoding for identifying plant species in two biodiversity hotspots: Mesoamerica and southern Africa. The researchers collected over 1,600 plant samples and tested eight potential DNA barcodes to determine which is most suitable for species identification. They identified a portion of the plastid *matK* gene as a universal DNA barcode for flowering plants due to its adequate rate of variation, ease of amplification, and alignment. They also demonstrated the applicability of DNA barcoding for biodiversity inventories, particularly in identifying cryptic species and those listed in the Convention on International Trade in Endangered Species (CITES). The study found that while the *trnH-psbA* region also performs well, its molecular evolution is complex, making it less reliable than *matK*. The *matK* gene was found to be the most suitable barcode, as it exhibits a "barcoding gap" between intra- and interspecific variations, allowing for clear species discrimination. The researchers also tested the utility of *matK* in identifying cryptic species, such as the orchid *Lycaste cf. tricolor*, which was found to be distinct from the typical *L. tricolor* based on morphological and genetic differences. The study highlights the importance of DNA barcoding in biodiversity conservation, particularly in regions with high species diversity and taxonomic challenges. It also underscores the need for further research to identify complementary barcodes and to improve the identification of difficult lineages. The results suggest that DNA barcoding with *matK* alone or in combination with *trnH-psbA* can achieve over 90% correct species identification, making it a valuable tool for biodiversity inventories and conservation efforts. The study concludes that *matK* is a preferred universal DNA barcode for flowering plants, with *trnH-psbA* serving as an alternative or complementary barcode.