Background: Cryptosporidium is an intracellular apicomplexan parasite that infects a wide range of vertebrates including humans. Cryptosporidiosis is a major cause of diarrhea in children with and without human immunodeficiency virus (HIV) infection in developing countries. More recently, the molecular methods for identification of morphologically indistinguishable species have been developed. The aim of this study was to determine the characterization of various species of this coccidian among children with diarrhea by using molecular methods. Methods: Fecal samples were collected from 1263 children with diarrhea who referred to Pediatrics Medical Centers in Qazvin and Tehran, two central provinces of Iran. Initial identification of Cryptosporidium was carried out by Zeihl-Neelsen acid-fast staining method of stool samples. DNA was extracted from positive microscopically samples and were subjected to a two step nested PCR-RFLP based on SSU-rRNA gene. Results: Out of 1263 collected samples, 31 (2.5%) were found to be contained Cryptosporidium oocysts. RFLP analysis showed that 80.6% of the positive isolates were Cryptosporidium parvum, 16.1% C. hominis and 3.2% had mix infection pattern of both C. parvum and C. hominis. Conclusion: Our results showed that the zoonotic pattern of transmission is predominant and has considerable significance in epidemiology of cryptosporidiosis in the study areas.