Cochlosoma anatis has been implicated in poult enteritis and mortality syndrome of turkey poults. The potential role of houseflies, Musca domestica L., in the epidemiology of this pathogen was investigated by examining the prevalence of C. anatis in houseflies collected from two Arkansas poultry farms. A 1520 bp region of C. anatis mtDNA 16S gene was subjected to DNA sequencing and a 486 bp portion was compared with other protozoan 16S sequences to develop PCR primers specific for C. anatis. Houseflies detected carrying C. anatis using the diagnostic 374 bp amplicons represented the first record of this protozoan in houseflies. This technique was also used to detect C. anatis from turkey gut samples and feces. This PCR diagnostic method allowed identification of C. anatis from houseflies, turkey gut, and fecal samples within 6 h after field-collected samples reached the laboratory.