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Background  Several Multilocus Sequence Typing (MLST) schemes have been developed for  Chlamydia trachomatis . Bom’s MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and  ompA . In contrast to other Chlamydia MLST schemes, Bom’s MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom’s MLST scheme in clinical samples with low concentrations of Chlamydia DNA.    Results  In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom’s MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested  in-silico  using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The  improved MLST  version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv.    Conclusions  The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of  Chlamydia  DNA. A similar concept could be applicable to other MLST schemes.    Supplementary Information  The online version contains supplementary material available at 10.1186/s12866-020-02077-y.

High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA