σ²⁸ RNA polymerase is an alternative RNA polymerase that has been proposed to have a role in late developmental gene regulation in Chlamydia, but only a single target gene has been identified. To discover additional σ²⁸-dependent genes in the Chlamydia trachomatis genome, we applied bioinformatic methods using a probability weight matrix based on known σ²⁸ promoters in other bacteria and a second matrix based on a functional analysis of the σ²⁸ promoter. We tested 16 candidate σ²⁸ promoters predicted with these algorithms and found that 5 were active in a chlamydial σ²⁸ in vitro transcription assay. hctB, the known σ²⁸-regulated gene, is only expressed late in the chlamydial developmental cycle only, and two of the newly identified σ²⁸ target genes (tsp and tlyC_1) also have late expression profiles, providing support for σ²⁸ as a regulator of late gene expression. One of the other novel σ²⁸-regulated genes is dnaK, a known heat shock-responsive gene, suggesting that σ²⁸ RNA polymerase may be involved in the response to cellular stress. Our σ²⁸ prediction algorithm can be applied to other bacteria, and by performing a similar analysis on the Escherichia coli genome, we have predicted and functionally identified five previously unknown σ²⁸-regulated genes in E. coli.