Streptococcus suis causes disease in both pigs and humans. Many virulence genes, including mrp, ef, sly, gapdh, fbps, and hyl, are shown to contribute to S. suis pathogenesis. In this study, the differential expression of the key S. suis serotype 2 virulence-associated genes was monitored 24 and 48 h after infection of HeLa cells. Gene expression was further investigated in bacteria harvested from the blood, lungs, brains, and synovia of swine 24, 48, and 72 h postintravenous infection with S. suis serotype 2. Bacterial RNA was extracted and gene expression was monitored using real-time reverse transcriptase-PCR at various time points. The results showed that after 24 h postinfection in vivo, mrp, gapdh, fbps, and hyl were highly expressed in all organs. At 48 h postinfection, ef and sly were highly expressed in the lungs and brain. At 72 h postinfection, all genes had a high level of expression in all organs. This study provides direct evidence that S. suis serotype 2 key virulence-associated genes are differentially expressed in a time-specific or an organ-specific manner in vivo. These findings also show the dynamic distribution of gene expression. Characterization of the differential expression patterns of this subset of key virulence-associated genes facilitates further study into the importance of these genes in S. suis serotype 2 pathogenesis in pigs.