Bacterial small regulatory RNAs (sRNAs) function in post-transcriptional control of gene expression and control a variety of processes including metabolic reactions, stress responses and pathogenesis in response to environmental signals. A variety of approaches have been used previously to identify 44 sRNAs in the opportunistic human pathogen Pseudomonas aeruginosa. In this work, RNA sequencing (RNA-seq) is used to identify novel transcripts in P. aeruginosa involving a combination of three different sequencing libraries. Almost all known sRNAs and over 500 novel intergenic sRNAs are identified with this approach. Although the use of three libraries increased the number of novel transcripts identified, there were significant differences in the subset of transcripts detected in each library, underscoring the importance of library preparation strategy and relative sRNA abundance for successful sRNA detection. Nearly 90% of the novel sRNAs have no orthologous bacterial sequences outside of P. aeruginosa, supporting a limited degree of sequence conservation and rapid evolution of sRNAs at the species level. We anticipate that the data will be useful for the study of regulatory sRNAs in bacteria and that the approach described here may be applied to identify sRNAs in any bacterium under different growth and stress conditions.