Restriction fragment length polymorphisms (RFLPs) of rRNA genes of Chlamydia spp. were analyzed. A Southern analysis of chromosomal DNA digests with cloned rRNA gene probes revealed the presence of one locus for rRNA genes on chromosomal DNAs in Chlamydia psittaci, Chlamydia pecorum, and Chlamydia pneumoniae and two loci in Chlamydia trachomatis. The RFLPs of rRNA genes were characteristic for each Chlamydia sp. DNA probes cloned from flanking regions of the rRNA genes of avian C. psittaci hybridized either with only C. psittaci or with only the avian and ovine abortion strains of C. psittaci. The RFLPs of rRNA genes and the flanking regions provide suitable genetic markers for differentiation of Chlamydia spp.