CtHtrA is a serine protease and chaperone that likely functions in the cell envelope of Chlamydia for protein assembly, maintenance, and degradation during stress response. Previously we demonstrated that CtHtrA is essential for the replicative phase of growth by the identification and application of a specific inhibitor. The objective of this study was to isolate and characterise mutants with resistance to JO146 by the generation of an EMS mutation library in C. trachomatis strain D. The C. trachomatis D mutation library was generated and confirmed using frequency of rifampicin resistance. The libraries of mutants (and a control that was not mutated) were subjected to selection protocols. JO146 resistant mutants were plaque purified from separate libraries and characterised genotypically and phenotypically. The selection resulted in three independent mutants with reduced susceptibility to JO146. The three mutants all had different non-synonymous variants in CT776 a acyl-ACP synthetase (AasC), and two of them had separate mutations (one being a null) in CT206 a predicted acyl-transferase. The likely (unconfirmed) role of the two loci is in the lipid fatty acid composition by acquisition/integration of fatty acids from the host cell. The mutants produced more infectious progeny than the wild-type, but several other phenotypic traits (persistence, heat stress) were not distinguishable from the wild-type. The results may indicate that either the inhibitor activity or HtrA activity is functionally linked to the composition of the lipid bilayer.