Background: The lack of engineered C. trachomatis (CT) genetic mutant libraries continues to hamper our understanding of the underlying mechanisms of CT pathogenesis as well as the evaluation of live attenuated strains for vaccine potential. Here, we report the development of a targeted mutagenesis system for CT. This technological platform utilizes the lambda red homologous recombination system and enables the rapid construction of CT mutants with specific gene deletions. Methods: We designed a vector that encodes the lambda red genetic elements (gam, exo, and beta) under regulation of an inducible promoter, a selection cassette containing chloramphenicol acetyl transferase and GFP, homologous flanking sequences (~2 kb) for targeting the insert to chromosomal regions of interest, and flippase recognition target (FRT) sites for downstream marker removal. For the initial development of this system, the CT L2/434 strain was chosen as a target strain, and mutants were selected and propagated in McCoy cells under multiple rounds of chloramphenicol selection. Results: As a proof of principle, we used the system to delete the CT virulence factor IncA. We obtained incA deletion mutants within 2 rounds of CT growth, and the resulting clones (CTΔincA) were verified by PCR-based genotyping, immunofluorescence microscopy using a specific antibody to IncA, and functionally by phenotypic assessment of cells infected with CTΔincA. We next used the technology to target 4 additional genomic loci hypothesized to play important roles in CT pathogenesis and virulence: incDEFGA (5 genes), cdsZ, dub1dub2 (2 genes), and pmpE. Mutant CT strains were obtained and genetically confirmed for each of these targets. The ease of generating mutant clones for these 4 target loci highlights the versatility and efficiency of the lambda red system for CT, and we predict the system can be easily applied to other Chlamydia species, for example C. muridarum which is widely used for the mouse model of upper genital tract infection and immunological studies. Conclusion: The potential applications of this technology are vast and include the ability to study the virulence factors of Chlamydia, develop new drug targets, and explore vaccine candidates. [ABSTRACT FROM AUTHOR]