Chlamydophila abortus (C. abortus) is one of the most important infectious agents causing abortion in ruminants. The bacterium is obligately intracellular, cannot grow on agar, but it needs cell culture or embryonated eggs for growth. Therefore, culture-independent detection methods such as the polymerase chain reaction are increasingly important and needed. The aim of this study was to develop a polymerase chain reaction assay with an internal control for detection of C. abortus in clinical samples. Using newly-designed two primer sets specific for C. abortus, the polymerase chain reaction assay was first tested with positive and negative control DNA and its sensitivity and specificity were determined. A new polymerase chain reaction protocol was developed by combining the new primer pair sets with bovine (12SM-FW and 12SBT-REV2) and ruminant host-specific primer sets (12S-FW and 12S-REV). In conclusion, the developed polymerase chain reaction assays can potentially be used for direct detection of Chlamydophila abortus in bovine and ruminant samples.