Several genotypic and phenotypic microbial source tracking (MST) methods have been proposed and utilized to differentiate groups of microorganisms, usually indicator organisms, for the purpose of tracking sources of fecal pollution. Targeting of host-specific microorganisms is one of the approaches currently being tested. These methods are useful as they circumvent the need to isolate individual microorganisms and do not require the establishment of reference databases. Several studies have demonstrated that the presence and distribution of Enterococcus spp. in feces seems to be influenced by the host species. Here, we present a method for detection of genetic sequences in culturable enterococci capable of identifying human sources of fecal pollution in the environment. The human fecal pollution marker designed in this study targets a putative virulence factor, the enterococcal surface protein (esp), in Enterococcus faecium. This gene was detected in 97% of sewage and septic samples but was not detected in any livestock waste lagoons or in bird or animal fecal samples. Epidemiological studies in recreational and groundwaters have shown enterococci to be useful indicators of public health risk for gastroenteritis. By identifying the presence of human fecal pollution, and therefore the possible presence of human enteric pathogens, this marker allows for further resolution of the source of this risk.