Giardia duodenalis has a wide genetic variety, and its characterization helps in the understanding of its transmission dynamics and in the development control strategies. This study aimed to assess the genetic diversity of G. duodenalis obtained in different Brazilian biomes and estimate their phylogenetic relationships. Three surveys including 944 participants were carried out in the municipalities of Russas (RSS, Caatinga semiarid biome), Santa Isabel do Rio Negro (SIRN, Amazon rainforest biome) and Nossa Senhora de Nazaré (NSN, Cerrado-Caatinga transition biome). G. duodenalis-positive fecal samples were submitted to amplification of gene fragments encoding β-giardin (βG, N = 71), glutamate dehydrogenase (GDH, N = 42), and triosephosphate isomerase (TPI, N = 27). Overall detection rates of assemblage A in G. duodenalis-positive samples through βG, GDH and TPI were 22/71 (31%), 13/42 (31%), and 13/27 (48.1%), respectively. Concerning assemblage B, rates with distinct genetic markers were 49/71 (69%), 29/42 (69%), and 14/27 (51.9%), respectively. In the Amazon, assemblage B was more prevalent (77.8%, 71.8% and 65% through βG, GDH and TPI, respectively), while in the Cerrado biome assemblage A predominated (50%, 66.6%, and 85.7%, through βG, GDH and TPI, respectively). In Caatinga biome assemblage A also predominated (71.4%, through βG). Thirty new sub-assemblages are described for assemblage B (24 βG and six TPI), as well as three new sub-assemblages are described for assemblage A (one GDH and 2 TPI). Higher genetic diversity of assemblage B in the Amazon may be related to demographic concentration leading to a more complex transmission network within a poorer sanitation background. The high genetic divergence between assemblages A and B (5.5-6.3%) support the proposal of taxon separation in distinct species.