Many strains of Chlamydia suis, a pathogen of pigs, express a stable tetracycline resistance phenotype. We demonstrate that this resistance pattern is associated with a resistance gene, tet(C), in the chlamydial chromosome. Four related genomic islands were identified in seven tetracycline-resistant C. suis strains. All resistant isolates carry the structural gene tet(C) and the tetracycline repressor gene tetR(C). The islands share significant nucleotide sequence identity with resistance plasmids carried by a variety of different bacterial species. Three of the four tet(C) islands also carry a novel insertion sequence that is homologous to the IS605 family of insertion sequences. In each strain, the resistance gene and associated sequences are recombined into an identical position in a gene homologous to the inv gene of the yersiniae. These genomic islands represent the first examples of horizontally acquired DNA integrated into a natural isolate of chlamydiae or within any other obligate intracellular bacterium.