Murray Valley encephalitis virus (MVEV) is a zoonotic flavivirus endemic to Australia and Papua New Guinea. A recent outbreak of MVEV resulted in four human cases and two deaths has prompted renewed concerns regarding the potential for MVEV to generate disease outbreaks. Phylogenetic analyses with earlier and recent circulating MVEV isolates may provide insights into virus evolution and genetic diversity. To date, there are only nine unique full genome MVEV sequences publicly available. The Ralph Doherty virus collection located at QIMR Berghofer MRI houses a significant number of virus isolates from field studies dated between the 1950s-1980s. The collection contained 53 unique virus isolates previously classified as MVEV. We recovered and grew and sequenced these MVEV isolates to expand the number of full length genomic sequences available for this virus. Of the stored MVEV isolates, 43 generated cytopathic effects (CPE) in BHK cells, and after RNA-Seq analysis, 30 unique MVEV sequences emerged. Phylogenetic analyses was performed with these isolates and publicly available full genome (n=9), pre-membrane (prM) and envelope (E) (n=66), and non-structural (NS) proteins (n=9) MVEV isolates over a 67 year period. Analyses indicated distinct clustering of the four recognised genotypes of MVEV, with 29 MVEV isolates from the Doherty collection clustering with GI and one isolate clustering with GIII. Sequence alignments revealed a number of unique amino acids between the different genotypes, with a number of conservative and non-conservative changes observed across prM, E and NS proteins. In vitro characterisation of selected isolates from GI, GII and GIII showed CPE in cell lines including neural progenitor, porcine and mammalian cells. Establishment of MVEV mouse models and structural work of the untranslated regions is currently ongoing. Altogether, this may provide insights into the diversity of MVEV for informing future intervention design.