High-resolution screening methodologies which enable the differentiation of Chlamydia trachomatis at the strain level, directly from clinical samples, can provide the detailed information required for epidemiological questions such as the dynamics of treatment failure. In addition, they give a detailed snapshot of circulating C. trachomatis genetic variation, data which are currently lacking for the Australian population. In the context of two Australian clinical trials, we assessed the genetic diversity of C. trachomatis and compared these to strains circulating globally. We used high-resolution multilocus sequence typing (MLST) of five highly variable genetic regions of C. trachomatis to examine variation in Australia. Samples with established genovars were drawn from a pool of 880 C. trachomatis-positive samples from two clinical studies, whereby 76 sample pairs which remained C. trachomatis-positive for the same genovar after treatment underwent MLST analysis to distinguish between treatment failure and reinfection. MLST analysis revealed a total of 25 sequence types (STs), six new allele variants and seven new STs not described anywhere else in the world, when compared to those in the international C. trachomatis MLST database. Of the eight most common global STs, seven were found in Australia (four derived from men who have sex with men (MSM) and three from heterosexuals). Newly identified STs were predominantly found in samples from the MSM population. In conclusion, MLST provided a diverse C. trachomatis strain profile, with novel circulating STs, and could be used to identify local sexual networks to focus on interventions such as testing and partner notification to prevent reinfection.