Recently, we reported the global regulatory roles of the two small RNAs in Salmonella typhimurium, ryhB-1 and ryhB-2. However, the genetic basis of the sRNA-target interactions remains unknown. To identify the nucleotides of RyhB-1 that are functionally important for its regulatory actions, we introduced random single-point mutations into ryhB-1 gene on the chromosome of Salmonella typhimurium carrying a sodB-lacZ translational fusion by an error-prone PCR method. We reasoned that mutants expressing variant RyhB-1 with weakened interaction with sodB transcript would produce a higher level of SodB when compared to wild type, leading to darker blue colonies on X-gal agar plates. Five mutants displaying a significant increase in beta-galactosidase activity under the condition inducing RyhB-1 expression were isolated. Quantitative real-time PCR analysis showed that the expression levels of eight target mRNAs in these five mutants were significantly changed when compared to the parent strain. Interestingly, two mutations affected growth and cell survival under H2O2-stressed conditions. The results suggest that there are strong selective constraints against mutational changes in ryhB-1 gene sequence, leading to high levels of nucleotide conservation in ryhB-1 gene sequences among the genus of Salmonella.