Genomic screens for small RNA candidates in Enterobacteriacae genomes were carried out with existing small RNA sequences, conserved flanking genes, and genomic backbone information. The small RNA sequences and contexts from E. coli K12 formed the basis of the search. Sequence identity identified 117 additional small RNA homologs in related genomes. Motifs of continuous sequence stretches added another 48 sRNA regions, termed partial homologs. However, this study is unique in identifying 160 nonhomologous sRNA loci in related genomes based on the conserved flanking gene synteny and the backbone retention information obtained from KEGG-SSDB. Gene synteny and genomic backbone continuity were observed to be correlated with all of the sRNAs in related genomes. This search is the first of its kind toward identification of functionally important regions using gene order and back-bone information. A disruption in flanking gene order or genomic backbone indicates a possible hotspot for alien gene pool integration. This study reports both occurrence of multiple copies of a sRNA and co-occurrence of different sRNAs between a pair of conserved flanking genes. In general, synteny and genomic backbone retention information can be added as additional search criteria toward the design of precise bioinformatics tools for sRNA, gene identification, and gene functional annotations in related genomes.