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Phytotherapeutic preparations from Uncaria guianensis (Aubl.) J.F. Gmel. (Rubiaceae) are marketed worldwide and are mainly used for their anti-inflammatory activity. The species has not yet been domesticated and is threatened by deforestation and overexploitation. It is, therefore, important to preserve and manage this genetic resource in germplasm banks, so that the extractive provision of plant material can be replaced by cultivated production. The aim of this study was to evaluate the genetic diversity among 20 genotypes maintained under in vitro conditions using 9 primers start codon targeted (SCoT) polymorphism, and to determine the concentrations of the pentacyclic oxindole alkaloids (POAs); mitraphylline and isomitraphylline in methanolic extracts by high-performance liquid chromatography (HPLC). Plantlets were cultivated on woody plant medium supplemented with 20 g.L−1 sucrose and 4.4 μM benzylaminopurine and incubated under a 16 h photoperiod for 45 days. SCoT analysis separated the genotypes into four divergent clusters and confirmed significant genetic diversity with up to 70% dissimilarity. Moreover, HPLC revealed considerable chemical variability and allowed the separation of the tested genotypes into high, medium and low producers of mitraphylline/isomitraphylline. Genotypes with the highest concentrations of POAs originated from the state of Acre and Amapá, while those with the lowest levels were from the state of Pará. The results demonstrate that the genetic diversity within the in vitro germplasm bank is sufficient to support breeding studies, selection of elite genotypes and the large-scale multiplication of plants that could serve as feedstock for the industrial-scale production of phytomedicines. [ABSTRACT FROM AUTHOR]