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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 …