DNA barcoding is a technique that uses short DNA sequences to identify species, aiding in ecological and conservation studies where traditional taxonomy is impractical. While established in animals, a universal plant barcode remains controversial. This study evaluated eight potential plant barcodes using over 1,600 samples from two biodiversity hotspots: Mesoamerica and southern Africa. The matK gene portion of the plastid genome was identified as a suitable universal barcode for flowering plants due to its adequate variation, ease of amplification, and alignment. It also demonstrated applicability in biodiversity inventories, particularly in identifying cryptic species and CITES-listed orchids. The study found that matK exhibited the highest interspecific divergence and the lowest intraspecific divergence, making it suitable for DNA barcoding. However, the trnH-psbA region also performed well, though its molecular evolution is complex. Both matK and trnH-psbA were effective in recovering species as monophyletic, with matK showing slightly better results. Combining these loci provided only moderate improvements in species identification. The study also highlighted the importance of DNA barcoding in identifying cryptic species, such as Lycaste tricolor, which showed distinct morphological and genetic differences. The matK gene was found to be easily amplified and aligned, making it a preferred universal barcode. The trnH-psbA region, while useful, was more challenging to align due to length variation and pseudogene presence. The results indicate that matK is the most suitable barcode for flowering plants, with trnH-psbA as an alternative or complementary barcode. DNA barcoding with matK alone or in combination with trnH-psbA can significantly aid in biodiversity inventories and conservation efforts, particularly in identifying CITES-listed species. The study emphasizes the need for DNA banking strategies to ensure long-term benefits of DNA barcoding efforts and to support future complementary barcodes. Overall, the study supports the use of matK as a preferred universal DNA barcode for flowering plants.DNA barcoding is a technique that uses short DNA sequences to identify species, aiding in ecological and conservation studies where traditional taxonomy is impractical. While established in animals, a universal plant barcode remains controversial. This study evaluated eight potential plant barcodes using over 1,600 samples from two biodiversity hotspots: Mesoamerica and southern Africa. The matK gene portion of the plastid genome was identified as a suitable universal barcode for flowering plants due to its adequate variation, ease of amplification, and alignment. It also demonstrated applicability in biodiversity inventories, particularly in identifying cryptic species and CITES-listed orchids. The study found that matK exhibited the highest interspecific divergence and the lowest intraspecific divergence, making it suitable for DNA barcoding. However, the trnH-psbA region also performed well, though its molecular evolution is complex. Both matK and trnH-psbA were effective in recovering species as monophyletic, with matK showing slightly better results. Combining these loci provided only moderate improvements in species identification. The study also highlighted the importance of DNA barcoding in identifying cryptic species, such as Lycaste tricolor, which showed distinct morphological and genetic differences. The matK gene was found to be easily amplified and aligned, making it a preferred universal barcode. The trnH-psbA region, while useful, was more challenging to align due to length variation and pseudogene presence. The results indicate that matK is the most suitable barcode for flowering plants, with trnH-psbA as an alternative or complementary barcode. DNA barcoding with matK alone or in combination with trnH-psbA can significantly aid in biodiversity inventories and conservation efforts, particularly in identifying CITES-listed species. The study emphasizes the need for DNA banking strategies to ensure long-term benefits of DNA barcoding efforts and to support future complementary barcodes. Overall, the study supports the use of matK as a preferred universal DNA barcode for flowering plants.