Karyotypic analysis of six species of the Australian leptodactylid frog genus Neobatrachus showed that N. pictus, N. centralis, N. pelobatoides and N. wilsmorei are diploid (2n = 24) while N. sudelli and N. sutor are tetraploid (4n = 48). Polyploidy has not been reported previously among Australian anurans. Idiograms of the six species indicate that they are similar to the other Australian leptodactylids so far discribed. DNA values of the tetraploids are approximately double the values for diploids. Tetraploid nuclear and cell sizes are greater compared with diploids but total body size shows no increase. At diakinesis in primary spermatocytes of tetraploids, mainly tetravalents together with a few bivalents are present. Silver straining of metaphase spreads clearly demonstrates the location of NORs at the secondary constrictions and their frequent association in the tetraploid N. sutor. Nucleolar number in interphase nuclei provides a reliable guide for distinguishing tetraploid from diploid frogs in the absence of chromosome analysis and can be determined for both living and preserved specimens. The possible origins and relationships of the tetraploid species are discussed.