Background: As the importance of chlamydial infections in pigs has become more obvious, a rapid and sensitive method to study the prevalence of Chlamydia suis in pig herds is required. Such a method should permit routine diagnostic tests for herds with clinical and subclinical infections, without the need for Chlamydia culture. Results:The main objective of this study was to develop a specific and rapid method for detecting C. suis in swine herds. A real-time PCR assay using a single locked nucleic acid (LNA)-containing probe specific for C. suis was developed based on the previously described 28S rDNA fragment used to identify Chlamydiales. Use of LNA nucleotides enabled the single probe to target a short, specific fragment of the 23S rRNA. The probe showed high specificity for C. suis and did not show any cross-reactivity with other Chlamydia or Chlamydophila species nor with swine DNA. All of the 86 tested field isolates, earlier identified as C. suis, were confirmed as positive using the newly developed assay. Conclusions: Using single LNA-based C. suis-specific probe allowed rapid and simple identification of this pathogen without requiring sequencing analysis and culturing. The proposed method may be used to study the prevalence of C. suis infection in pig herds and as a routine diagnostic test for herds with clinical and subclinical infection.