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The organisms investigated are members of the Hypsolebias flavicaudatus species complex; a clade of morphologically similar annual killifishes endemic to the semi-arid savannah of north-eastern Brazil in the Sao Francisco River basin, which is a tropical area under intensive process of habitat loss. A taxonomic revision was conducted combining two different approaches for species delimitation - a tree-based approach using mt-DNA (cytochrome b) and a character-based approach using morphological variation (colour patterns, fin morphology, meristic and morphometric data). Nine species are recognised, of which five are new species: H. flagellatus (Costa), H. flavicaudatus (Costa & Brasil), H. gilbertobrasili sp. nov., H. guanambi (Costa & Amorim), H. janaubensis (Costa), H. nitens sp. nov., H. pterophyllus sp. nov., H. radiseriatus sp. nov. and H. sertanejo sp. nov. On the basis of 17 years of field studies, preliminary conservation status assessments indicate that two species are highly threatened with extinction, at least one is endangered and another is vulnerable, whereas the others are not endangered. This result deeply contrasts with a previous assessment 10 years ago, when the whole H. flavicaudatus complex was considered as a single species of no conservation concern. Discrepancies in the assessment of conservation among closely related species living in similar habitats are a consequence of heterogeneous human occupation and disproportionate recent development throughout the Sao Francisco River basin.