The genetic relatedness of ten Murray Valley encephalitis virus (MVE) isolates from Australia has been examined by comparing HaeIII and TaqI restriction digest profiles of cDNA to virion RNA. The isolates were from the Murray Valley region of south-eastern Australia and from the Ord River region of Western Australia and spanned a period of 23 years (1951-1974). The isolates generated closely similar restriction digest profiles. The extent of similarity suggested that the level of nucleotide sequence divergence between any pair of Australian MVE isolates is probably around 1%. The genetic homogeneity of the MVE isolates contrasts with results obtained for Ross River virus, an alphavirus, using an identical methodological approach; we propose that this difference results from the important role of birds in the life cycle of MVE. Four MVE isolates from three fatal human cases showed small genetic differences one from the other. These isolates did not have a common restriction digest profile which distinguished them from strains obtained from other sources (e.g., from mosquitoes or a heron). The data do not support the view that clinical cases of MVE infection in humans are due to a particular strain of virus although this has not been rigorously excluded. The two available MVE isolates from Papua New Guinea (PNG) were from the Sepik and Port Moresby regions. They generated HaeIII and TaqI restriction digest profiles which were different both from each other and from those of the Australian type. Genetic divergence between the two PNG isolates was estimated to be approximately 6%; divergence between either of the PNG isolates and the Australian type was greater than 6%. Our data suggest that the evolution of MVE in Australia and PNG has proceeded independently and that circulating Australian MVE strains are not systematically re-seeded from regions of endemicity in PNG. Studies on the relatedness of MVE and two close antigenic relatives, Japanese encephalitis virus (JE) and Alfuy virus (ALF), showed that the genetic relatedness between any MVE isolate and JE or ALF is less than that between the most divergent of the MVE isolates, including those from Papua New Guinea.