Background: Current approaches to utilise self-replicating transposon-delivery vectors for transposon mutagenesis in Chlamydia spp. are limited by early background activation of the tetracycline-controlled transposase. This causes premature transposon induction and death of early transformants. Theophylline-controlled riboswitches, which regulate gene translation, may provide additional control over transposase expression and improved regulation of transposon induction. Methods: Two different riboswitch variants were cloned into a transposon-delivery vector for C. muridarum, via Hi-Fi DNA Assembly, and transformed into C. muridaurm Nigg P-. Transformed C. muridarum was used to infect McCoy cell monolayers for 16 hours post infection (hpi) after which the transposase was induced with theophylline and anhydrotetracycline to trigger transposon activity. At 24 or 40 hpi, the transposon library was harvested, DNA was purified and sequenced with Nanopore technology to identify transposon insertions. Results: Use of Riboswitch A produced 14 unique transposon insertions, while Riboswitch D resulted in 153 at 24hpi. Extending the induction period by harvesting at 40hpi increased the number of unique insertions detected with Riboswitch D to 505. Control experiments without theophylline or anhydrotetracycline showed background transposase expression in Riboswitch D, resulting in 50 and 63 unique insertions at 16 hpi and 40 hpi respectively. No transposon insertions were detected in the Riboswitch A control. Conclusions: This study expanded the number of transposon mutants that can be generated in Chlamydia and reports the largest number of transposon mutants to date altogether covering 39.5% of the genes, rRNA, and tRNA sequences in C. muridarum.