Repeated infections of Chlamydia trachomatis (CT) occur frequently in young adults. These may be new infections, or persistent infections due to treatment failure or unresolved infections in sex partners. We compared CT multilocus sequence typing (CT-MLST) to ompA genotyping in discriminating new from persistent Chlamydia infections. Samples from young heterosexual persons were selected from Dutch screening implementation studies in Amsterdam and Rotterdam, the Netherlands, between 2009 and 2011. Paired CT positive samples at baseline (T0) and after 6 months (T1) were genotyped with 6 MLST loci which included: ompA , CT046 , CT058 , CT144 , CT172 and CT682 . The uniqueness of Chlamydia strains was assessed by adding samples from 256 heterosexuals in Amsterdam. For 27 out of 34 persons with repeated infections, full MLST types were obtained for paired samples. In 17 of these 27 persons a multilocus (n = 13) or single locus variant (n = 4) was found, indicating new CT infections at T1. For 5 MLST discordant participants, the ompA genovar was identical. The 10 persons with concordant typing results were categorised as treatment failure (5 persons) or unresolved infections (5 persons). A minimum spanning tree, generated from all cases and 256 reference samples showed large and small clusters and singletons. Surprisingly, the persons with concordant samples had CT strains that were either unique (singleton) or found in small clusters. The median time between T0 and T1 did not differ between the persons with concordant and discordant samples. The number of sex partners before T0 however, was higher for the discordant group. High resolution sequence typing was superior compared to ompA typing in discriminating new from persisting Chlamydia infections. Many persons (37%) showed exactly the same Chlamydia strain after 6 months indicating possible treatment failure.