BACKGROUND: Chlamydia trachomatis infection is one of the most common sexually transmitted infections (STIs). This study investigated the prevalence and genotype distribution of C. trachomatis, and treatment response, focusing on the recent emergence of genovariant L2b in Taiwan. METHODS: This prospective study was conducted from August 2021 to September 2023, enrolling 1023 participants, including 492 people who received pre-exposure prophylaxis (PrEP) for HIV and 531 people with HIV (PWH). Multiple-site sampling and genotyping of C. trachomatis identified were performed. Participants testing positive for C. trachomatis received a 7-day course of doxycycline and test-of-cure (TOC) assessments were conducted post-treatment. RESULTS: Among the participants, mostly MSM (92.7%), 26.4% tested positive for C. trachomatis, with 77.8% identified in the rectum. The prevalent genotypes were J (26.4%), G (24.0%), and B (17.7%). Treatment with a 7-day course of doxycycline resulted in clearance for most cases (91.1%). Genotyping investigations for those repeatedly testing positive (8.9%) showed reinfections with different genotypes. Eighteen cases of asymptomatic rectal carriage of genovariant L2b were detected, predominantly in PWH (88.9%). The L2b genovariant was cleared with 7 days of doxycycline on TOC assessments. The prevalence of L2b increased over time, with multi-locus sequence typing showing ST53 as the predominant strain. CONCLUSIONS: C. trachomatis was prevalent among PWH and PrEP users and the prevalence of L2b genovariant was increasing in Taiwan. A 7-day course of doxycycline was effective in clearing L2b genovariant in asymptomatic participants. Continued surveillance to monitor the evolving epidemiology of chlamydia in Taiwan is warranted.