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• Isolation of Orthoflavivirus ilheussense (ILHV) in Psorophora (Janthinosoma) ferox collected in cerrado biome of Brazil. • This is the first report of the circulation of ILHV in the state of Goiás, an arbovirus associated with febrile illness, which is often accompanied by encephalitis. • The strain is closely related to ILHV isolated in Belém (PA) during the dry season. • The ILHV detection in various regions from Brazil indicates the risk of the emergence of new diseases. The Orthoflavivirus ilheusense (ILHV) is an arbovirus that was first isolated in Brazil in 1944 during an epidemiologic investigation of yellow fever. Is a member of the Flaviviridae family and it belongs to the antigenic complex of the Ntaya virus group. Psorophora ferox is the primary vector of ILHV and this study presents the isolation and phylogenetic analysis of ILHV in a pool of Ps. ferox collected in the state of Goiás in 2021. Viral isolation tests were performed on Vero cells and C6/36 clones. The indirect immunofluorescence test (IFI) was used to confirm the positivity of the sample. The positive sample underwent RT-qPCR, sequencing, and phylogenetic analysis. This is the first report of ILHV circulation in this municipality and presented close relationship between this isolate and another ILHV isolate collected in the city of Belém (PA). [Display omitted] [ABSTRACT FROM AUTHOR]