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The mid-north-eastern Caatinga is a semiarid freshwater ecoregion in North-eastern Brazil that is dominated by temporary rivers and is currently classified as one of the least ichthyologically-known ecoregions in the world. The present study aimed to provide an updated checklist of mid-north-eastern Caatinga ecoregion (MNCE) freshwater fish species and evaluate their taxonomic identity using morphology, DNA barcoding and multiple species delimitation approaches. After reviewing published studies and ichthyological collections, 119 species were identified. Among these were 94 putatively valid native and 14 non-native species, five undescribed native species, four new records for the MNCE, 11 potential cases of misidentification and 14 species listed as inquirenda. Additionally, 252 individuals from 49 species were barcoded, revealing three potential taxonomic synonyms. The combined molecular approaches estimated a total of 91 native species, although a finalized species list for the MNCE awaits additional taxonomic revisions and field surveys. This study provides the most up-to-date species checklist for the MNCE and a molecular reference database for identifying MNCE fishes with DNA barcodes. Results highlight the need to integrate traditional taxonomy with molecular approaches to correctly identify species, especially in taxonomically problematic ecoregions such as the MNCE., (© 2018 The Fisheries Society of the British Isles.)