Viral hemorrhagic septicemia virus (VHSV) has caused significant economic losses in the olive flounder (Paralichthys olivaceus) aquaculture industry in South Korea since the first reported outbreak in 2001. While horizontal transmission has been the primary focus in VHSV research, the potential for vertical transmission remains largely unexplored. In this study, we investigated whether VHSV can infect gonadal tissues of olive flounder and evaluated the possibility of vertical transmission. Following experimental infection, VHSV RNA was quantitatively detected in blood and mucus samples when the viral load in target tissues exceeded 3.16×10? copies/g, suggesting that these non-lethal samples may serve as useful materials for early diagnosis of infection. Notably, VHSV RNA was continuously detected in the ovaries up to 21 days post-infection, reaching a maximum of 2.19×10? copies/g and 6.31×10³ TCID??/mL. Furthermore, RNA in situ hybridization (RNA-ISH) revealed VHSV localization in the ovarian tissues, including vitellogenic oocytes, surrounding follicular cells, and ovigerous folds. These results indicate that VHSV can persist in the gonads of infected broodstock under low-temperature conditions (13℃ or lower), and suggest the potential for vertical transmission through infected ...