Routine genetic manipulation approaches are currently limited in Chlamydia. Here, Kokes et al. generated a collection of chemically mutagenized Chlamydia trachomatis strains and identified the induced single-nucleotide variants (SNVs) using whole-genome sequencing. The authors describe the use of this library in a microscopy-based forward genetic screen for mutants impaired in filamentous actin assembly at chlamydial inclusions, and they identified a role for the bacterial inclusion membrane protein InaC in proper actin localization at these sites. The authors also found that C. trachomatis tolerates nonsense mutations in distinct genes of the tricarboxylic acid cycle and the DNA damage response. Thus, this method could be a useful tool for reverse and forward genetic applications that can be applied to microorganisms that are not amenable to classic genetic manipulations.