Chlamydia trachomatis is the most common bacterial sexually transmitted infection worldwide and the leading cause of preventable blindness in developing countries. Tetracycline is commonly the drug of choice for treating C. trachomatis infections, but cases of antibiotic resistance in clinical isolates have previously been reported. Here, we used antibiotic resistance assays and wholegenome sequencing to interrogate the hypothesis that two clinical isolates (IU824 and IU888) have acquired mechanisms of antibiotic resistance. Immunofluorescence staining was used to identify C. trachomatis inclusions in cell cultures grown in the presence of tetracycline; however, only antibiotic-free control cultures yielded the strong fluorescence associated with the presence of chlamydial inclusions. Infectivity was lost upon passage of harvested cultures grown in the presence of tetracycline into antibiotic-free medium, so we conclude that these isolates were phenotypically sensitive to tetracycline. Comparisons of the genome and plasmid sequences for the two isolates with tetracycline-sensitive strains did not identify regions of low sequence identity that could accommodate horizontally acquired resistance genes, and the tetracycline binding region of the 16S rRNA gene was identical to that of the sensitive control strains. The porB gene of strain IU824, however, was found to contain a premature stop codon not previously identified, which is noteworthy but unlikely to be related to tetracycline resistance. In conclusion, we found no evidence of tetracycline resistance in the two strains investigated, and it seems most likely that the small, aberrant inclusions previously identified resulted from the high chlamydial load used in the original antibiotic resistance assays. © 2013 SGM.