Alphaviruses are responsible for a significant number of animal and human diseases. They are enveloped RNA viruses that cycle between invertebrate insect vectors and vertebrate hosts; for most alphaviruses, the vectors are mosquitoes. Alphavirus infection in humans can result in diseases characterized by fever, rash, arthritis or encephalitis. In Australia, the most common alphavirus diseases are caused by infection with Ross River virus (RRV) and Barmah Forest virus (BFV), and rarely, Sindbis virus (SINV); Chikungunya infection is often identified in travellers returning from the Asia Pacific region. We isolated a novel alphavirus from a pool of Culex annulirostris mosquitoes trapped in 2011 in Derby, a town in the Kimberley region of northern Western Australia. Preliminary phylogenetic analysis of the complete genome sequence has shown this virus, provisionally named Derby virus (DERV), sits within the Western equine encephalitis complex between Whataroa virus (the only endemic New Zealand alphavirus) and the Southwest genotype of Sindbis virus. We analysed acute phase serum samples collected from febrile individuals throughout Western Australia to determine anti-alphavirus neutralising antibody profiles, using a serum plaque reduction neutralisation test (PRNT). Sera were diluted and incubated with titrated DERV, RRV, BFV, and SINV, then inoculated onto Vero cells; presence of neutralising antibody was indicated by reduction of plaques on the Vero cell monolayer compared to serum-free virus control and PRNT titre calculated. Pools of diagnosed RRV+; BFV+; and RRV- and BFV- sera, 100 sera in total, were analysed. RRV+ and BFV+ sera neutralised their homologous viruses at high titres, as expected. Of the fifteen RRV- and BFV- pools, one demonstrated DERV PRNT-70 titres of >80, compared with RRV, BFV, and SINV which produced PRNT titres of 10 or less. Current work aims to assess the serological relationship of DERV to other SINV lineages; avian and human cell tropism; and to analyse more samples from febrile patients in Western Australia to begin assessing public health significance of infection with DERV.