Streptococcus agalactiae is a significant pathogen in Nile tilapia (Oreochromis niloticus) culture, causing economic losses in Thailand. Formalin-killed S. agalactiae vaccine in tilapia can be used in the prevention of diseases and mortalities. In this study, we administered a formalin-killed S. agalactiae vaccine to individual tilapia broodstock to activate immunoglobulin (IgM) gene expression. The IgM gene expression levels differed among individual broodstock and were grouped into four categories: high IgM males (HM), low IgM males (LM), high IgM females (HF), and low IgM females (LF). Using these categories, we designed matings to create four breeding groups: HM × HF, HM × LF, LM × HF, and LM × LF, generating three fry families per group. The tilapia fry was then orally challenged with S. agalactiae for 14 days. The results showed a significant difference (p<
0.05) in survival rates among the breeding groups. The HM × HF group had the highest survival at 93.33 ± 6.20%, while the LM × LF group had the lowest at 21.11 ± 6.50%. Gene expression analysis of toll-like receptor 2, hepcidin, immunoglobulin, MHC class II, and granzyme genes at 24, 48, 72, and 96 hours post S. agalactiae challenge revealed significant temporal changes. IgM and granzyme expression levels varied notably among groups, with IgM peaking at 72-96 hours and showing a significant difference (p<
0.05) between groups. The HM×HF, HM×LF, and LM×HF breeding groups displayed significantly higher IgM expression levels compared to the LM×LF group. Granzyme gene expression also showed significant differences (p<
0.05) from 48 hours post-challenge, with the highest expression observed in the HM×HF group and the lowest in the LM×LF group. No significant difference (p>
0.05) was observed between the HM×LF and LM×HF groups for granzyme expression. This experiment concludes that high levels of IgM gene expression of broodstock tilapia induced by formalin-killed S. agalactiae vaccine could be used as a gene marker to select the S. agalactiae-resistant tilapia in breeding program.