Functional genetic analysis of has been a challenge due to the historical genetic intractability of , although recent advances in chlamydial genetic manipulation have begun to remove these barriers. Here, we report the development of the Himar C9 transposon system for , a mouse-adapted species that is widely used in infection models. We demonstrate the generation and characterization of an initial library of 33 chloramphenicol (Cam)-resistant, green fluorescent protein (GFP)-expressing transposon mutants. The majority of the mutants contained single transposon insertions spread throughout the chromosome. In all, the library contained 31 transposon insertions in coding open reading frames (ORFs) and 7 insertions in intergenic regions. Whole-genome sequencing analysis of 17 mutant clones confirmed the chromosomal locations of the insertions. Four mutants with transposon insertions in , , , and were investigated further for and phenotypes, including growth, inclusion morphology, and attachment to host cells. The mutant was shown to be incapable of complete glycogen biosynthesis and accumulation in the lumen of mutant inclusions. Of the 3 mutants, was shown to have the most pronounced growth attenuation defect. This initial library demonstrates the utility and efficacy of stable, isogenic transposon mutants for The generation of a complete library of mutants will ultimately enable comprehensive identification of the functional genetic requirements for infection Historical issues with genetic manipulation of have prevented rigorous functional genetic characterization of the ∼1,000 genes in chlamydial genomes. Here, we report the development of a transposon mutagenesis system for , a mouse-adapted species that is widely used for investigations of chlamydial pathogenesis. This advance builds on the pioneering development of this system for We demonstrate the generation of an initial library of 33 mutants containing stable single or double transposon insertions. Using these mutant clones, we characterized phenotypes associated with genetic disruptions in glycogen biosynthesis and three polymorphic outer membrane proteins.