| Summary: | Abstract Background Accurate identification of the species of Anopheles Meigen, 1818 requires careful examination of all life stages. However, morphological characters, especially those of the females and fourth-instar larvae, show some degree of polymorphism and overlap among members of species complexes, and sometimes even within progenies. Characters of the male genitalia are structural and allow accurate identification of the majority of species, excluding only those in the Albitarsis Complex. In this key, based on the morphology of the male genitalia, traditionally used important characters are exploited together with additional characters that allow robust identification of male Anopheles mosquitoes in South America. Methods Morphological characters of the male genitalia of South American species of the genus Anopheles were examined and employed to construct a comprehensive, illustrated identification key. For those species for which specimens were not available, illustrations were based on published illustrations. Photographs of key characters of the genitalia were obtained using a digital Canon Eos T3i attached to a light Diaplan Leitz microscope. The program Helicon Focus was used to build single in-focus images by stacking multiple images of the same structure. Results An illustrated key to South American species of Anopheles based on the morphology of the male genitalia is presented, together with a glossary of morphological terms. The male genitalia of type-specimens of previously poorly documented species were also examined and included in the key, e.g. Anopheles (Anopheles) tibiamaculatus (Neiva, 1906) which has a unique quadrangular-shaped aedeagus with an apical opening. Conclusions Male genitalia of South American species of Anopheles possess robust characters that can be exploited for accurate species identification. Distortion that can occur during the dissection and mounting process can obstruct accurate identification; this is most evident with inadvertent damage or destruction of unique features and interferes with correctly assigning shapes of the features of the ventral claspette. In some species, the shape, and anatomical details of the aedeagus also need to be examined for species identification. For members of the Myzorhynchella Series, both ventral and dorsal claspettes possess multiple characteristics that are herein used as reliable characters for species identification.
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