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Leaf-cutter bees (genus Megachile Latreille) are among the most common and diverse group of bees. However, the identity and taxonomic placement of many species are problematic and species identification is often difficult. Some species are known only from a single specimen or from one of the sexes, and identification keys are not available for many groups. We address these taxonomic issues for the subgenera Rhyssomegachile Mitchell and Zonomegachile Mitchell, two poorly known South American lineages of leaf-cutter bees. We provide comparative diagnoses, redescriptions, illustrated identification keys, new geographical records, and designate needed neotypes for Megachile cara Mitchell, M. gigas Schrottky, M. guayaqui Schrottky, M. reliqua Mitchell, M. sanctipauli Schrottky, M. stabilis Mitchell, and M. turbulenta Mitchell. We resurrect M. tricosa Cockerell from synonymy with M. urbana Smith and synonymize M. turbulenta under M. tricosa. We recognize four species in Rhyssomegachile and eight species in Zonomegachile. In the latter subgenus, we revalidate M. reliqua from synonymy with M. moderata and propose the following four new species: Megachile kalina, new species, from French Guiana; M. durantae, new species, from Rondônia, Brazil; M. paisa, new species, from Antioquia, Colombia; and M. uncinata, new species, from Catamarca, Argentina. We confirm sex associations in Zonomegachile and describe its nest for the first time. Megachile tricosa, M. ardua Mitchell, and M. tacanensis Moure, currently assigned to Rhyssomegachile, exhibit morphological features that do not fit any of the known subgenera. Thus, we use a cladistic analysis to explore their phylogenetic relationships and establish two new subgenera for these species: Aporiochile Gonzalez and Engel, new subgenus, for M. tricosa and Chalepochile Gonzalez and Engel, new subgenus, for the remaining two species. We provide an updated key to the subgenera of Megachile s.l. of the Western Hemisphere.