In order to improve the molecular resources available for conservation management of Madagascar's threatened ichthyofauna, we elaborated a curated database of 2860 mitochondrial sequences of the mitochondrial COI, 16S and ND2 genes of Malagasy fishes, of which 1141 sequences of freshwater fishes were newly sequenced for this data set. The data set is mostly composed of COI (2015 sequences) while 16S and ND2 sequences from partly the same samples were used to match the COI sequences to reliably identified reference sequences of these genes. We observed COI uncorrected pairwise genetic distances of 5.2‒31.0% (mean 20.6%) among species belonging to different genera, and 0.0‒22.4% (mean 6.4%) for species belonging to the same genus. Deeply divergent mitochondrial lineages of uncertain attribution were found among Malagasy freshwater eleotrids and gobiids, confirming these groups are in need of taxonomic revision. DNA barcodes assigned to introduced cichlids (tilapias) included Coptodon rendallii, C. zillii, Oreochromis aureus (apparently a new country record), O. cf. mossambicus, O. niloticus, and one undetermined species of Oreochromis, with sequences of up to three species found per location. In aplocheiloid killifishes of the genus Pachypanchax, most species from northern Madagascar had only low mitochondrial divergences, three of these species (P. omalonotus, P. patriciae, and P. varatraza) were not reciprocally monophyletic, and one genetically deviant lineage was discovered in a northern locality, suggesting a need for partial taxonomic revision of this genus. While the lack of voucher specimens for most of the samples sequenced herein precludes final conclusions, our first step towards a DNA barcoding reference library of Madagascar's fishes already demonstrates the value of such a data set for improved taxonomic inventory and conservation management. We strongly suggest further exploration of Madagascar's aquatic environments, which should include detailed photographic documentation and tissue sampling of large numbers of specimens, and collection of preserved voucher specimens as well as of living fish for the buildup of ex situ assurance populations of threatened species complying with the One Plan Approach proposed by the IUCN SSC Conservation Breeding Specialist Group (CBSG).