ObjectivesChlamydia trachomatisis classified into 15 major genotypes, A to L3, based on the diversity ofompAgene. Here, we evaluated and characterised the distribution and diversity ofompA-genotypes over 32 years (1990–2021) in Portugal.MethodsThe collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824C.trachomatis-positive samples that were successfullyompA-genotyped between 1990 and 2021. An in-depth analysis ofompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed.ResultsompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGVompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples.ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV casesperyear) and the non-negligible detection ofompA-genotype B in urogenital and anorectal specimens.ConclusionsThis study provides a comprehensive landscape ofC. trachomatismolecular surveillance in Portugal, highlighting the continued relevance ofompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding ofC. trachomatisepidemiology, diversity and pathogenicity.