Two subtractive complementary DNA libraries were constructed from Nile tilapia Oreochromis niloticus vaccinated with formalin-killed Streptococcus iniae cells, and a further two constructed from O. niloticus infected with S. iniae. Of the 68 distinct expressed sequence tag (EST) contigs and singletons, 45 and 13 EST shared high similarities with genes of known and unknown functions, respectively. Ten EST contigs and singletons had no significant similarity to any sequences. Five putative immune-relevant genes, β2m, α-ha, mmp9, pgrn and cxcr4, were selected for quantitative reverse-transcription polymerase chain reaction in four strains of Oreochromis spp.: genetically improved farmed tilapia (O. niloticus), Oreochromis aureus, O. niloticus and O. niloticus×O. aureus, with different disease resistance following infection with S. iniae. pgrn was up-regulated more significantly in disease-resistant strains than in the susceptible. α-ha was markedly down-regulated, and no significant differences in the expression level of β2m were detected. A negative correlation was observed between the expression of mmp9 and that of cxcr4. The results provide insight into the molecular response of O. niloticus to S. iniae infection.