Mariri or jagube (Banisteriopsis C. B. Rob.) is a vine of the Malpighiaceae family and is naturally found in the Brazilian Amazon. It is also an important ingredient of ayahuasca tea, which is used by some traditional, indigenous people and religious groups of Amazonian origin. Via DNA barcodes, this study aimed to evaluate the efficiency of six markers to access genetic diversity and use them in the identification of either ethnovarieties or species of Banisteriopsis used in the preparation of “ayahuasca” in the Amazon. The efficiency of three plastid genes (matK/trnK, ndhF, and rcbL) and three nuclear markers (MP3 and yage5 isolated in the present study and ITS—Internal Transcribed Spacer) were tested. For these six markers, a total of 4792 base pairs were sequenced. The plastid markers revealed that all the samples analyzed belong to the genus Banisteriopsis; however, they presented the lowest indices of genetic diversity and were not efficient for use in the identification of ethnovarieties. Nuclear DNA markers showed the highest levels of genetic diversity. The ITS showed the highest values of genetic distances between the identified lineages. This study demonstrates that the use of ITS for DNA barcoding was more efficient since the identified lineages showed correspondence with the ethnovarieties of Banisteriopsis spp. used in the preparation of ayahuasca. As such, we encourage new integrative studies like this one, which combines modern approaches and traditional knowledge, to contribute to the maintenance of traditional knowledge as well as the conservation of species.