Abstract Streptococcus agalactiae causes a severe systemic disease in fish, and the routes of entry are still ill‐defined. To address this issue, two groups of 33 red tilapia Oreochromis spp. each of 10 g were orally infected with S . agalactiae ( n = 30), and by immersion ( n = 30), six individuals were control‐uninfected fish. Three tilapias were killed at each time point from 30 min to 96 h post‐inoculation ( pi ); controls were killed at 96 h. Samples from most tissues were examined by haematoxylin–eosin (H&E), indirect immunoperoxidase ( IPI ) and periodic acid‐Schiff; only intestine from fish infected by gavage was evaluated by transmission electron microscopy. The results of both experiments suggest that the main entry site of S. agalactiae in tilapia is the gastrointestinal epithelium; mucus seems to play an important defensive role, and environmental conditions may be an important predisposing factor for the infection.