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The family Totiviridae contains non-enveloped, doublestranded RNA viruses with genomes of approximately 4.6–7 kbp in length. These totiviruses are classified into five genera: Totivirus, Victorivirus, Trichomonasvirus, Giardiavirus, and Leishmaniavirus (Nibert 2007). They are associated with latent infections of fungal or protozoan hosts (Li et al. 2020). Recently, a few totiviruses have been identified and shown to have unexpected host distributions, including insects, plants, and even Chromalveolata (Chen et al. 2016; Koyama et al. 2015; Marchler-Bauer et al. 2017; Mor and Phelps 2016). However, most of these new viruses have not been assigned to appropriate genera by the International Committee on Taxonomy of Viruses (ICTV). Omono River virus (ORV) is an unclassified RNA virus in the family Totiviridae (Isawa et al. 2011; Okamoto et al. 2016). In this study, we reported a novel ORV isolated from a pool of Culex tritaeniorhynchus mosquitoes collected in Yunnan, China. We also updated the phylogenetic relationships of the familyTotiviridae to include all genomes reported to date. Mosquitoes were collected using ultraviolet light lamps in Yunnan, China in 2012. The captured mosquitoes were grouped according to the location and mosquito classification. Approximately 200 mosquitoes in each group were milled together and cultured with Aedes albopictus C6/36 cells. After blind-passaging by three times, the pool that caused a cytopathic effects (CPE) was further selected for RNA extraction and deep sequencing on an S5 sequencer. We identified a new ORV from the culture products of the Culex tritaeniorhynchus pool collected in Yuanjiang County. This virus was designated as Omono River virus strain YJ according to the collection site. YJ could be stably passaged in C6/36 cells and cause a CPE (Fig. 1A-a, 1A-b). The growth curves were determined by real-time polymerase chain reaction using standard dsRNA. YJ had strong proliferative ability in C6/36 cells and reached a plateau on the third day of incubation (Fig. 1B). Transmission electron microscopy revealed that the viral particles were uniformly spherical particles approximately 25 nm in diameter (Fig. 1A-c, 1A-d). The virus particles we observed were smaller than those of previously reported viruses. (e.g., 40 nm for other ORVs and C. tritaeniorhynchus totivirus KL) (Li et al. 2020). The genome of YJ was 7614 nucleotides long and shared the highest identity (79% nucleotide similarity and 93.13% amino acid similarity) with the known ORV strain TB94 (GenBank Accession Number: KY264025). The complete genome of YJ was deposited in GenBank (Accession Number: MN296094). The sequence of YJ encoded two open reading frames (ORFs). ORF1 (nt 7–5067) of YJ encoded a 1687aa capsid protein, whereas ORF2 (nt 5322–7538) of YJ encoded a 739aa RNA-dependent RNA polymerase (RdRp). The two proteins were translated into fusion protein owing to Electronic supplementary material The online version of this article (https://doi.org/10.1007/s12250-020-00247-w) contains supplementary material, which is available to authorized users.