Molecular techniques using fungal DNA barcoding (ITS) and other markers have been key to identifying the biodiversity of different geographic areas, mainly in megadiverse countries. Here, we provide an overview of the fungal diversity in Brazil based on DNA markers of phylogenetic importance generated since 1996. We retrieved fungal sequences of ITS, LSU, SSU, tef1-α, β-tubulin, rpb1, rpb2, actin, chitin synthase, and ATP6 from GenBank using different field keywords that indicated their origin in Brazil. A total of 19,440 sequences were recovered. ITS is the most representative marker (11,209 sequences), with 70.1% belonging to Ascomycota, 18.6% Basidiomycota, 10.2% unidentified, 1.1% Mucoromycota, two sequences of Olpidium bornovanus (Fungi incertae sedis), one sequence of Blastocladiomycota (Allomyces arbusculus), and one sequence of Chytridiomycota (Batrachochytrium dendrobatidis). Considering the sequences of all selected markers, only the phyla Cryptomycota and Entorrhizomycota were not represented. Based on ITS, using a cutoff of 98%, all sequences comprise 3047 OTUs, with the majority being Ascomycota (2088 OTUs) and Basidiomycota (681 OTUs). Previous numbers based mainly on morphological and bibliographical data revealed 5264 fungal species from Brazil, with a predominance of Basidiomycota (2741 spp.) and Ascomycota (1881 spp.). The unidentified ITS sequences not assigned to a higher taxonomic level represent 1.61% of all ITS sequences sampled and correspond to 38 unknown class-level lineages (75% cutoff). A maximum likelihood phylogeny based on LSU illustrates the fungal classes occurring in Brazil.