Aim: To design a rapid diagnostic test to differentiate species belonging to the family Chlamydiaceae. Methods and Results: Five oligonucleotide sets each targeting various conserved regions of the genome of six species (Chlamydia muridarum, C. suis, C. trachomatis, Chlamydophila felis, Cp. pneumoniae and Cp. psittaci) belonging to the family Chlamydiaceae were tested for their suitability for polymerase chain reaction (PCR) and high resolution melt (HRM) curve analysis to differentiate Chlamydiaceae species. Three of the oligonucleotide sets were able to detect all six reference species used in this study, but only one set (16SG) could clearly differentiate between them by HRM curve analysis. The PCR-HRM curve analysis confidence percentages correlated strongly with the nucleotide sequence identities. Clinical specimens from a number of animal species suspected of chlamydiosis were tested with the newly developed 16SG PCR-HRM curve analysis and sequenced to confirm the infecting species. It was demonstrated that PCR-HRM using the 16SG oligonucleotide set could relate the infecting Chlamydiaceae species to the most similar (based on 16S rRNA gene nucleotide sequence) reference species tested. Although Cp. pecorum was not included initially as a reference species in this assay, inclusion of a field isolate of Cp. pecorum as a reference allowed two koala specimens to be correctly identified. Conclusion: PCR-HRM analysis using the oligonucleotide set 16SG is a robust, simple and rapid technique for differentiation of at least the Chlamydiaceae species used in this study. Significance and Impact of the Study: This technique allowed for the rapid detection and identification of the six Chlamydiaceae reference species and may be useful for identification of uncharacterized Chlamydiaceae species or for use in animal species where occurrence of the disease has not been fully investigated.