Karyotypes of three microhylid frog species of the Malagasy relict genus Scaphiophryne were studied: Scaphiophryne gottlebei, S. madagascariensis and S. spinosa. The latter two showed a plesiomorphic ranoid karyotype of 2n = 26. In contrast, tetraploidy was demonstrated in S. gottlebei, which constitutes an exceptional state among Malagasy amphibians. A combination of different banding techniques and of rDNA-FISH provided evidence for allopolyploidy in the species and for a completed subsequent functional and structural diploidization. Phylogenetic analysis of mitochondrial 16S rDNA sequences revealed a significant deceleration of nucleotide substitution rates in Scaphiophryne. The tetraploidy of S. gottlebei probably occurred early in their radiation. Ecological and behavioural patterns of Scaphiophryne probably favoured intraspecific gene flow and hybridization events, thereby leading to slow molecular substitution rates and to allopolyploid chromosome speciation in S. gottlebei.