W ith ≈15,000 laboratory-confirmed cases over the last decade, Barmah Forest virus (BFV) is the second most common cause of human arboviral disease in Australia, after Ross River virus (RRV) (1). BFV is a positive-sense, single-strand, enveloped RNA virus of genus Alphavirus, family Togaviridae. Other viruses in this genus include chikungunya virus, RRV, Sindbis virus, and Eastern and Western equine encephalitis viruses. BFV was first isolated in 1974 from Culex annulirostris mosquitoes trapped near Barmah Forest, northern Victoria, Australia (2); the first case of a clinical BFV infection in humans was reported in 1986 (3). Since then, BFV has been reported throughout mainland Australia and Papua New Guinea (4,5). Clinical signs and symptoms of BFV infection, including polyarthritis, arthralgia, and myalgia, are similar to but milder than those of RRV infection (6-7). Through phylogenetic analyses of the nucleotide sequences of complete E2 envelope protein genes and of the 3′ untranslated region (3′ UTR), we identified 3 BFV lineages. However, we found only 1 example of 2 of the lineages (5,8). RRV caused epidemic polyarthritis outbreaks in military personnel in Australia during and after short military exercises in the Shoalwater Bay Training Area in northeastern Australia in 2016 and 2017 (9). The soldier in this study was among personnel who sought treatment during the 2017 outbreak with a suspected RRV infection. Signs and symptoms included rash on the face and body, nausea, headache, fatigue, lethargy, and joint and muscle pain. This retrospective study was approved by the Australian Department of Defence and Department of Veterans' Affairs Human Research Ethics Committee (DDVA HREC), Joint Health Command Low-Risk Ethical Review Panel (no. 16-021). We obtained formal written consent from the soldier. During a retrospective investigation of the outbreak, using PanBio ELISA kits (Abbott, https://www.abbott. com), we detected BFV IgG and IgM, but not RRV IgG and IgM, in convalescent serum samples collected 23, 28, and 38 days after onset of symptoms in the patient. After inoculating 100 µL of the serum into cultures of C6/36 mosquito cells and 2 subsequent passages in this cell line, we did not detect infectious virus in the acutephase serum sample collected on the day of symptom onset. However, we detected BFV RNA, but not RRV RNA, using a quantitative reverse-transcription PCR assay of RNA extracted from 140 µL of the acute-phase sample using a QiaAMP Viral RNA Mini Kit (QIA-GEN, https://www.qiagen.com). BFV E2 RNA was present at 4.2 ×10 6 copies/mL, with a forward primer 8985F (5′-AGTGTGGCAGTACAACTCCCAAT-3′) corresponding to genome position 8985-9006 and a reverse primer (5′-AAGGCACATGGATCTTTCCTTTC-3′) corresponding to genome position 9036-9058. For sequencing, we amplified the E2 and 3′ UTR genes by reverse transcription PCR using primers E2 forward 8205F 5′-GCTGTCTGACCACTACTACCA-3′ and E2 reverse 9833R 5′-GACTTAATCACTACTA-AAGATAGCG-3′, and 3′ UTR forward 10923F 5′-TC-CATCCATCTCTACTACCG-3′ and reverse poly-T