Abstract: Cryptosporidium is an opportunistic parasite typically associated with large waterborne outbreaks. Surface waters contaminated with human and animal feces serve as main source for epidemic spread of Cryptosporidium parasites. In this study, we used a small-subunit rRNA-based PCR-Restriction Fragment Length Polymorphism (RFLP) technique to determine the prevalence and to characterize human-infective species of Cryptosporidium parasites in water samples collected from a stream in Ardabil city in Iran. Among 30 samples examined, 11 samples showed positive results. Restriction pattern analysis showed C. andersony as the most common species with 7 cases; followed by C. parvum, bovine genotype, with 3 cases and C. suis with 1 case. The results indicated that PCR-RFLP technique provides an applicable and feasible method for detection and identification of Cryptosporidium oocysts in environmental water samples. The results, furthermore, demonstrated that wildlife is the major source of Cryptosporidium oocysts in surface water resources in the study region.