The aim of this study was to isolate and characterize Clostridium tertium from chicken feces. C. tertium is found in soil as well as gastrointestinal tracts of humans and other animals. C. tertium is an anaerobic, motile, gram-positive, rod-shaped bacterium that forms highly resistant endospores without generating an exotoxin. Although it is considered an uncommon pathogen and its pathogenicity is often unclear, this bacterium has been implicated in bacteremia, pneumonia, enterocolitis, septic arthritis and abscess in humans and animals. Even though there is no direct evidence that C. tertitum can be transmitted from animals to humans, it is of importance to see the prevalence and distribution of C. tertium in animal feces. Chicken feces samples were collected from the research farm at Dankook University, South Korea. Ten chickens per cage were housed together and 10 chicken droppings per cage (a total of 10 cages) were collected using sterile disposable culture loops starting when chickens were 1 week of age then at one-week interval until they were 4 weeks of age. After ethanol treatment (50% ethanol for 1 h), dropping samples were streaked onto TCCFA agar plates with 5% sheep blood, and incubated in anaerobic jars at 37°C for 72 h. The isolates were identified based on morphological criteria. Then identification was confirmed by the Gram staining and sequencing of the V1-V3 of 16S rRNA gene. After 72 h incubation in an anaerobic jar, a pure culture of colonies was recovered. A Gram stained smear of colonies showed weak-staining, gram-positive rods. These rod-shaped bacteria were detected from all chicken feces. The cloning and subsequent sequencing of the 16S rRNA genes from 2 representative isolates identified them as C. tertium with a probability of 99%. To our knowledge, this is the first isolation of C. tertium from chicken droppings. In depth studies on pathogenesis of C. tertium will be needed to better understand potential roles of C. tertium in poultry health.