In April and May 2025, outbreaks of equine influenza occurred for the first time in 17 years in Japan. Equine influenza virus (EIV) of the H3N8 subtype was mainly detected in heavy draft horse populations in Kumamoto Prefecture and the Tokachi area of Hokkaido. In total, 10 EIVs were isolated from infected horses and then were used for genetic and serological analyses. Phylogenetic analysis of all eight genes revealed that all Japanese isolates were clustered with the Florida sublineage clade 1 (Fc1) viruses and were closely related to North American Fc1 viruses detected in 2024-2025. The results suggest that the epidemic virus was introduced from North America to Japan. The high sequence identity across all eight genes in these Japanese isolates indicates that EIVs from a common origin spread in Kumamoto Prefecture and the Tokachi area of Hokkaido. These two places are separated by a straight-line distance of approximately 1500 km, but draft horses are regularly transported between the two places and infected horses may have moved before movement restrictions were imposed. The virus neutralization assay showed that the horse antisera against A/equine/South Africa/4/2003 (a Fc1 vaccine strain recommended by the World Organisation for Animal Health) and A/equine/Ibaraki/1/2007 (a Fc1 vaccine strain in Japan) cross-neutralized well with two viruses isolated in Kumamoto Prefecture and the Tokachi area of Hokkaido in 2025. This suggests that the current Fc1 vaccine strains are effective against the epidemic Fc1 isolates in Japan in 2025.