Streptococcus suis is an important swine pathogen that can cause a variety of diseases. Streptococcus suis serotype 9 (SS9) is a prevalent serotype, but limited information is available. Here, we studied and compared 30 SS9 isolates, including 24 isolates from China between year 2004 and 2013, 5 isolates from Vietnam and a serotype reference isolate from Denmark. A multilocus sequence typing (MLST) analysis was performed to exploit the genetic relationships between those isolates. The phylogenetic tree based on the MLST data divides those isolates into two clades (I and II), revealing different evolutionary paths of collected strains. A virulence genotyping analysis was performed by detecting 23 virulence-related genes; the clustering result also revealed two clusters (I and II), highly in accordance with MLST analysis result, showing that phylogenetic relatedness led to the presence of similar virulence genotypes. Murine model infection experiment was performed to assess the virulence of those strains in cluster I and cluster II, which displayed high virulence diversity even within a same cluster/ST. Notably, ST 243 could be regarded as an ST with high virulence potential in SS9. In conclusion, this study has revealed high genetic and virulence diversity in SS9 isolates.