Amanita is a worldwide-distributed fungal genus, with approximately 600 known species. Most species within the genus are ectomycorrhizal (ECM), with some saprotrophic representatives. In this study, we constructed the first comprehensive phylogeny including ECM species from Colombia collected in native Quercus humboldtii forests and in introduced Pinus patula plantations. We included 8 species (A. brunneolocularis, A. colombiana, A. flavoconia, A. fuligineodisca, A. muscaria, A. rubescens, A. sororcula, and A. xylinivolva) out of 16 species reported for the country, two new reports: A. citrina and A. virosa, and a new variety A. brunneolocularis var. pallida. Morphological taxonomic keys together with a phylogenetic approach using three nuclear gene regions: partial nuc rDNA 28S nuc rDNA internal transcribed spacers ITS1 and ITS2 and partial translation elongation factor 1-α gene (TEF1), were used to classify the specimens. Several highly supported clades were obtained from the phylogenetic hypotheses obtained by Bayesian inference and maximum likelihood approaches, allowing us to position the Colombian collections in a coherent infrageneric level and to contribute to the knowledge of local Amanita diversity.