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The distribution and public health significance of Cryptosporidium species and genotypes in humans and bovine differ across geographical areas. Cryptosporidium species causes a disease known as cryptosporidiosis in humans and animals. To characterize the prevalence of cryptosporidiosis in humans in southern Assam, India, stool samples ( n = 1119) of diarrhea patients were collected from different hospitals and from the community during the period January 2014 to July 2016. Fecal smears were examined microscopically for Cryptosporidium species using modified acid fast staining and were screened to ascertain the presence of Cryptosporidium antigen by enzyme-linked immunosorbent assay (ELISA). The genomic DNA of positive fecal samples were analyzed by nested polymerase chain reaction (PCR), which were subsequently genotyped by PCR-restriction fragment length polymorphism (RFLP), based on small subunit (SSU) 18S rRNA. It was found that the prevalence of Cryptosporidium spp. was high during the monsoon season. The average infection rate of Cryptosporidium spp. was found to be 2.4% (27/1119) microscopically. When subjected to nested PCR using amplification of the 18S rRNA gene, Cryptosporidium was found to be 8.57% (98/1119). Based on the 18S rRNA gene, two Cryptosporidium spp., namely Cryptosporidium andersoni (6.97%: 78/1119) and Cryptosporidium parvum (1.7%: 20/1119), were identified. Cryptosporidium andersoni infections were found to be of either zoonotic or anthroponotic origin. The prevalence was statistically significant ( p = 0.03, R = 0.042) considering age, gender, and cast. [ABSTRACT FROM AUTHOR]