It has become generally accepted that the genus Cryptosporidium is composed of a number of host-adapted species and genotypes, with relatively few species or genotypes causing the majority of clinical disease in humans. Water is considered an important vehicle of transmission for Cryptosporidium and a large emphasis has been placed on monitoring the occurrence and prevalence of Cryptosporidium in water. The US Environmental Protection Agency's Method 1623 is a commonly used and accepted method for quantifying the number of Cryptosporidium oocysts in water. However, in light of molecular advancements that have led to a better understanding of the host-adapted nature of Cryptosporidium, basic quantification of oocyst concentrations in water provide inadequate information for assessing the environmental and public health risks associated with contamination of water sources with these parasites. A method of molecular forensic profiling has been developed as an extension of Method 1623 to provide identification of the species and genotypes of Cryptosporidium in water samples. Following quantification of oocysts by microscopy according to Method 1623, the slide material becomes the template for molecular analysis. The method consists of a replicated nested PCR-RFLP assay targeted against the 18S small ribosomal sub-unit RNA gene locus, with verification of RFLP patterns by DNA sequence analysis. One of the strengths of the method is the ability to resolve single genotypes from mixed populations of Cryptosporidium spp. that may be commonly found in environmental water samples. Molecular forensic profiling by this method has provided important information regarding the host-sources impacting a watershed and the health risks associated with these contamination events; information that will be critical in designing new models of public health protection of drinking water supplies based on comprehensive environmental risk assessments and source-to-tap watershed management policies. Includes 48 references, tables, figures.