The family Chlamydiaceae contains two genera and nine species. Rapid and easy identification of these species is essential for taxonomic, epidemiological and clinical determinations. Currently, DNA sequence analysis is the only accepted method that decisively distinguishes all nine species. In this study, a simple and rapid PCR-RFLP procedure was developed by which laboratory-cultured chlamydial specimens could be identified. To accomplish this, conserved oligonucleotide primers and restriction sites were deduced from 16S and 23S rRNA sequence data from > 50 chlamydial strains representing all nine species. DNA from 25 previously characterized chlamydial strains were tested with these primers and restriction enzymes. All nine chlamydial species were reliably distinguished in the tests. The procedure was optimized by adjusting the annealing temperature using both a standard and a heat-activated DNA polymerase to reduce mismatch PCR amplification of mycoplasmas and other bacteria. The result was that a PCR method for species identification of chlamydial isolates and for distinguishing mycoplasmas and chlamydiae was created. This method can be used to rapidly identify known species of the family Chlamydiaceae.