Fecal pollution of surface waters is a top reason for impairment, as reported in the U.S. Environmental Protection Agencys report on the quality of the Nations waters. To be able to develop and implement TMDLs for impaired aquatic resources, it is imperative to determine the sources of the contamination. One tool used to determine the sources of bacterial fecal contamination is to apply a microbial source tracking approach to the system of interest. Microbial source tracking (MST) approaches are based on the assumption that specific strains of bacteria, genetic fingerprints, or DNA-based markers are associated with specific host species. Because accurate source identification of fecal contamination is essential in MST, more sensitive, selective and reliable molecular markers are required. The two types of genotypic methods that have been applied widely in a variety of environments can be classified as library-independent (LI) and library-dependent (LD). For both types, the temporal and spatial stability of selected genotypes are aspects that need to be evaluated, and these aspects are often times missing when applying MST to environmental samples. LD-MST methods require the development of large databases comprised of source-specific isolates. Once a source-specific fingerprint has been identified, the temporal and spatial variability of that particular genotype still needs to be validated. LI-MST is based on the application of culture-independent methods such as amplification of DNA from environmental samples using 16S rDNA markers in combination with polymerase chain reaction (PCR).