Small noncoding RNAs (sRNAs) function as regulatory elements in both eukaryotes and bacteria. Trans-acting bacterial sRNAs posttranscriptionally regulate gene expression by base pairing with target mRNAs, which often leads to changes in translation efficiency and/or stability of the transcript. Bioinformatic search algorithms along with a variety of experimental approaches have become increasingly useful for the discovery of sRNAs and their mRNA targets. Our laboratory and others recently demonstrated that Hfq, a protein chaperone of sRNAs in bacteria, is required for the full virulence of both Yersinia pestis, the bacterium that causes the disease plague, and the genetically related gastrointestinal pathogen Yersinia pseudotuberculosis. This led us to pursue the first global identification and analysis of sRNAs in pathogenic Yersinia species. We have identified 150 previously unannotated sRNAs expressed by Y. pseudotuberculosis when cultured in vitro at either 26°C or 37°C, the majority of which are Yersinia-specific. The deletion of multiple Yersinia-specific sRNAs from either Y. pseudotuberculosis or Y. pestis leads to the attenuation of these pathogens in mouse models of infection. In addition, we have identified the mRNA targets controlled by one of these virulence-associated sRNAs, suggesting potential new virulence determinants in Y. pseudotuberculosis.