Uncariaspecies are used in traditional medicine and are considered of high therapeutic value and economic importance. This work describes the assembly and annotation of the chloroplast genomes ofU. guianensisandU. tomentosa, as well as a comparative analysis. The genomes were sequenced on MiSeq Illumina, assembled with NovoPlasty, and annotated using CHLOROBOX GeSeq. Addictionaly, comparative analysis were performed with six species from NCBI databases and primers were designed in Primer3 for hypervariable regions based on the consensus sequence of 16 species of the Rubiaceae family and validated on an in-silico PCR in OpenPrimeR. The genome size ofU. guianensis and U. tomentosawas 155,505 bp and 156,390 bp, respectively. Both Species have 131 genes and GC content of 37.50%. The regionsrpl32-ccsA, ycf1,andndhF-ccsAshowed the three highest values of nucleotide diversity within the species of the Rubiaceae family and within theUncaria genus, these regions weretrnH-psbA,psbM-trnY,andrps16-psbK.Our results indicates that the primer of the regionndhAhad an amplification success for all species tested and can be promising for usage in the Rubiaceae family. The phylogenetic analysis recovered a congruent topology to APG IV. The gene content and the chloroplast genome structure of the analyzed species are conserved and most of the genes are under negative selection. We provide the cpDNA of NeotropicalUncariaspecies, an important genomic resource for evolutionary studies of the group.