There are many gaps to be filled in our understanding of mosquito-borne viruses, their relationships with vectors and reservoir hosts, and the environmental drivers of seasonal activity. Stratford virus (STRV) belongs to the genus Flavivirus and has been isolated from mosquitoes and infected humans in Australia but little is known of its vector and reservoir host associations. A total of 43 isolates of STRV from mosquitoes collected in New South Wales between 1995 and 2013 was examined to determine the genetic diversity between virus isolates and their relationship with mosquito species. The virus was isolated from six mosquito species; Aedes aculeatus, Aedes alternans, Aedes notoscriptus, Aedes procax, Aedes vigilax, and Anopheles annulipes. While there were distinct differences in temporal and spatial activity of STRV, with peaks of activity in 2006, 2010 and 2013, a sequence homology of 95.9%-98.4% was found between isolates and the 1961 STRV prototype with 96.2%-100% identified among isolates. Temporal differences but no apparent nucleotide divergence by mosquito species or geographic location was evident. The result suggests the virus is geographically widespread in NSW (albeit only from coastal regions) and increased local STRV activity is likely to be driven by reservoir host factors and local environmental conditions influencing vector abundance. While STRV may not currently be associated with major outbreaks of human disease, with the potential for urbanisation and climate change to increase mosquito-borne disease risks, and the possibility of genomic changes which could produce pathogenic strains, understanding the drivers of STRV activity may assist the development of strategic response to public health risks posed by zoonotic flaviviruses in Australia.