The emergence of antibiotic‐resistant pathogenic strains of Lactococcus garvieae serotype II isolated from fish in Japan has become a growing concern in recent years. The data on drug susceptibility and its associated resistance mechanism are limited. Therefore, the present study was conducted to determine the minimum inhibitory concentrations (MICs) of chemotherapeutic agents against 98 pathogenic strains of emerging Lactococcus garvieae serotype II isolated from fish from six different prefectures in Japan from 2018 to 2021. The tested strains were resistant to erythromycin, lincomycin and tiamulin. PCR amplification revealed the presence of erm(B) in all erythromycin‐resistant strains, while a conjugation experiment confirmed that these strains carried erm(B) that could be transferred to recipient Enterococcus faecalis OG1RF with frequencies from 10−4 to 10−6 per donor cells. Nucleotide sequencing of the representative isolated plasmid pkh2101 from an erythromycin‐resistant strain showed that it was a 26,850 bp molecule with an average GC content of 33.49%, comprising 31 CDSs, 13 of which remained without any functional annotation. Comparative genomic analysis suggested that pkh2101 shared the highest similarity (97.57% identity) with the plasmid pAMbeta1, which was previously isolated clinically from Enterococcus faecalis DS‐5. This study provides potential evidence that the plasmid harbouring erm(B) could be a source of antibiotic resistance transmission in emerging L. garvieae infection in aquaculture.