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We provide a DNA barcoding survey of Malagasy amphibians, including 251 of the 292 nominal species known to date, by complementing previous data with 280 newly determined barcoding sequence fragments of the mitochondrial cytochrome oxidase subunit I (COI) gene. Amplification success for the newly determined sequences was highest (94%) with one set of universal COI primers (dgLCO1490-dgHCO2198) while other primer sets had distinctly lower success rates. By and large, we observed relatively high average interspecific genetic distances of 25-27% within the Mantellidae and Microhylidae, and genetic distances of 13-21% within the Hyperoliidae. Lower values of 6-7% were observed between some sister species in all families, with extreme lows of 0.2-0.3% between a few sister species pairs in microhylids and mantellids for which we postulate mitochondrial introgression or yet unsettled taxonomy. Within-species divergences were relatively high especially in mantellids where they averaged 5.3%, due to the inclusion of numerous deep conspecific lineages (by definition with high divergences to other specimens) in our study. Above this, the degree of polymorphism was difficult to establish owing to limited sampling per population in our assessment. Compared to a previous assessment from 2009 based on 16S rDNA sequences, we identify 14 additional undescribed candidate species and raise the maximum estimate of species in the island’s batrachofauna to well over 500.