Over the past 15 years, microbial functional genomics has been made possible by the combined power of genome sequencing and microarray technology. However, we are now approaching the technical limits of microarray technology, and microarrays are now being superseded by transcriptomics based on high-throughput (next generation) DNA-sequencing technologies. The term RNA-seq has been coined to represent transcriptomics by next-generation sequencing. Although pioneered on eukaryotic organisms due to the relative ease of working with eukaryotic mRNA, the RNA-seq technology is now being ported to microbial systems. This review will discuss the opportunities of RNA-seq transcriptome sequencing for microorganisms, and also aims to identify challenges and pitfalls of the use of this new technology in microorganisms.