Cyclospora cayetanensis is an intestinal coccidian protozoan that has emerged as an important cause of both epidemic and endemic protracted diarrhea worldwide. Though humans appear to be the only natural hosts; the role of animals as natural reservoir is uncertain but of increasing concern. The present study aimed to study the prevalence of coccidian in different groups such as immunocompromised, clinically apparent immunocompetent and healthy individuals. Also, the study isolates were assessed for heterogeneity among the sequences.Stool samples from different groups of patients were collected. The parasite was detected in stool by different diagnostic tools such as light microscopy and nested PCR-restriction fragment length polymorphism using 18S ribosomal RNA as the target gene.The prevalence of C. cayetanensis was 2.4% (19/800) in the present study. The PCR assay amplified Cyclospora cayetanensis DNA in only 89% (17/19) isolates. Further, sequencing revealed no significant difference among the study isolates and the non-primates. Phylogenetic analysis of the study isolates however, formed two clusters. While one cluster showed close evolutionary association with the C. cayetanensis strains, the other cluster showed evolutionary association with the two non-primate species.The methods described here for detection of C. cayetanensis oocysts are simple, efficient, specific, and sensitive and therefore can be effectively applied for laboratory diagnosis and environmental assessment of fresh produce and water sources. Clinicians should include Cyclospora infection in the differential diagnosis of prolonged or relapsing diarrheal illness even in clinically apparent immunocompetent individuals.