Abstract Phytoplasmas are bacteria transmitted by insects that can cause plant diseases. In Bogotá ' Candidatus Phytoplasma asteris' and ' Candidatus Phytoplasma fraxini', infect 11 species of urban trees, weeds, grass, potato and strawberry. A set of primers, that amplify both phytoplasmas species were designed and used for absolute and relative qPCR quantification of the 16SrRNA gene. The primers AJ-16Sr-F/AJ-16Sr-R allowed the amplification of ‘ Ca. P. asteris’, ‘ Candidatus Phytoplasma palmae’, ‘ Ca. P. fraxini’ and ‘ Candidatus Phytoplasma phoenicium’, not of ‘ Candidatus Phytoplasma pruni’. Absolute qPCR detected phytoplasmas between 1 × 10 9 and 1 × 10 3 copies/μL DNA extract. Two species-specific hydrolysis probes, AJ-16SrI-Cy5.5 and AJ-16SrVII-TexRed, were designed to detect ' Ca . P. asteris' and ' Ca . P. fraxini' respectively, using the AJ-16Sr-F/AJ-16Sr-R primers. For relative quantification, the 18SrRNA gene was used as normalizer. Relative qPCR detected phytoplasmas between 1 × 10 9 and 1 × 10 3 copies/μL DNA extract. Multiplex reactions allowed the specific quantification of ' Ca . P. asteris', ' Ca . P. fraxini' in comparison to the normalizer. qPCR methods were validated on natural hosts Andean oak trees and leafhoppers . The relative quantification values were higher for ' Ca . P. fraxini' (x̅ RQ = 3203.1 ± 2622,9 n = 14) compared with ' Ca . P. asteris' (x̅ RQ = 14.9 ± 24,5 n = 6) in oak tree samples. In the leafhoppers, the relative quantification values ranged between RQ = 26.5 and RQ = 294,927.3 for ' Ca . P. fraxini’ and RQ = 34.8 and RQ = 1722.2 for ' Ca . P. asteris'. In conclusion, although absolute qPCR allowed the quantification of phytoplasmas by comparing Cq (quantification cycle) values of samples with a standard curve, it did not allow to differentiate between ' Ca . P. asteris' and ' Ca. P. fraxini'. In contrast, relative qPCR assays using specific hydrolysis probes allowed the specific detection and quantification of each phytoplasma, in individual and mixed infections in insect vectors and plant hosts.