Aim: To identify a DNA sequence specific to a bacterium found in poultry litter that was indicative of faecal contamination by poultry sources. Methods and Results: Faecally contaminated poultry litter and soils were used as source material for the development of a quantitative polymerase chain reaction (qPCR) method targeting the 16S rRNA gene of a Brevibacterium sp. The identified sequence had 98% nucleotide identity to the 16S rRNA gene of Brevibacterium avium. The qPCR method was tested on 17 soiled litter samples; 40 chicken faecal samples; and 116 nontarget faecal samples from cattle, swine, ducks, geese, and human sewage collected across the United States. The 571-bp product was detected in 76% of poultry-associated samples, but not in 93% of faecal samples from other sources. Marker concentrations were 107-109 gene copies per gram in soiled litter, up to 105 gene copies per gram in spread-site soils, and 107 gene copies per litre in field run-off water. Results were corroborated by a blinded study conducted by a second laboratory. Conclusion: The poultry-specific PCR product is a useful marker gene for assessing the impact of faecal contamination as a result of land-applied poultry litter. Significance and Impact of the Study: This study describes the first quantitative, sensitive and specific microbial source tracking method for the detection of poultry litter contamination.