Pentatrichomonas hominis is considered a commensal protozoan in the large intestine of a number of mammalian hosts, such as cats, dogs, and non-human primates. The resulting infections, which can induce diarrhea, have been attributed to opportunistic overgrowth of P. hominis. This study was performed to confirm the P. hominis infection and its molecular characterization from the feces of puppies with diarrhea. Fecal samples were obtained from 14 German shepherd puppies with diarrhea over 1 week (7 females and 7 males, 2-9 months of age) residing on a dog farm in August 2007. Species-specific PCR assay identified P. hominis 18S rRNA genes in 3 of the 14 puppies (1 female and 2 males; 1 aged 2 months and 2 aged 9 months). This phylogenetic analysis established that P. hominis belonged to the 1st clade, which is comprised of Bos taurus and Felines.