Murray Valley encephalitis virus (MVEV) is a medically important mosquito-borne flavivirus found in Australia and Papua New Guinea (PNG). Partial envelope gene nucleotide sequences of 28 isolates of MVEV from Western Australia (WA) between 1972 and 2003 were aligned and compared phylogenetically with the prototype MVE-1-51 from Victoria in 1951 and isolates from northern Queensland and PNG. Monoclonal antibody-binding patterns were also investigated. Results showed that the majority of isolates of MVEV from widely disparate locations in WA were genetically and phenotypically homogeneous. Furthermore, isolates of MVEV from WA and northern Queensland were almost identical, confirming results from earlier studies. Recent isolates of MVEV from Western Province in PNG were more similar to Australian isolates of MVEV than to isolates from PNG in 1956 and 1966, providing further evidence for the movement of flaviviruses between PNG and Australia. Additional representatives of a unique variant of MVEV (OR156) from Kununurra in the northeast Kimberley region of WA were also detected. This suggests that the OR156 lineage is still intermittently active but may be restricted to a small geographic area in northern WA, possibly due to altered biological characteristics.