Twelve captive magnificent tree frogs Litoria splendida and 2 green tree frogs L. caerulea on a property in the Darwin rural area (Northern Territory, Australia) either died or were euthanased after becoming lethargic or developing skin lesions. Samples from both species of frog were submitted for histopathology and virus isolation. An irido-like virus was cultured from tissue samples taken from both species and was characterised using electron microscopy, restriction enzyme digests and nucleic acid amplification and sequencing. The isolates were determined to belong to the genus Ranavirus, were indistinguishable from each other and shared a 98.62% nucleotide similarity and a 97.32% deduced amino acid homology with the Bohle iridovirus over a 1161 bp region of the major capsid gene. This is the first isolation of a ranavirus from amphibians in the Northern Territory and the first report of natural infection in these 2 species of native frog. The virus is tentatively named Mahaffey Road virus (MHRV). Dis Aquat Org. 2012, 99: 169–177. R. P. Weir, N. J. G. Moody, A. D. Hyatt, S. Crameri, R. Voysey, J. Pallister, I. V. Jerrett. Department of Resources, PO Box 3000, Darwin, Northern Territory 0800, Australia. richard.weir@nt.gov.au