To investigate the prevalence of Cyclospora cayetanensis in a longitudinal study and to conduct a population genetic analysis, fecal specimens from 6579 patients were collected during the cyclosporiasis – prevalent seasons in two urban areas of central China in 2011–2015. The overall incidence of C. cayetanensis infection was 1·2% (76/6579): 1·6% (50/3173) in Zhengzhou and 0·8% (26/3406) in Kaifeng (P < 0·05), with infections in all age groups (P > 0·05). All the isolates clustered in the C. cayetanensis clade based on the small subunit ribosomal RNA gene sequence phylogenetic analysis. There were 45 specimens positive for all the five C. cayetanensis microsatellite loci, and formed 29 multilocus genotypes (MLGs). The phylogenetic relationships of 54 distinct MLGs (including 25 known reference MLGs), based on the concatenated multilocus sequences, formed three main clusters. A population structure analysis showed that the 79 isolates (including 34 known reference isolates) of C. cayetanensis produced three distinct subpopulations based on allelic profile data. In conclusion, we determined the frequency of C. cayetanensis infection in humans in Henan Province. The clonal population structure of the human C. cayetanensis isolates showed linkage disequilibrium and three distinct subpopulations.