To improve our understanding of the genetic diversity of the human-infecting coccidium, Cyclospora cayetanensis, 160 clinical fecal samples representing Canadian cyclosporiasis cases from four provinces (ON=119, QC=24, BC=7, NL=10) identified between 2010 and 2020, were genotyped using a next generation sequencing targeted amplicon approach consisting of eight markers. Genotyping data were collected for at least one marker for 96.2% (154/160) of specimens and only 36.9% (59/160) of specimens had genotyping data for all eight markers. We identified eighteen genetic clusters from the 79.4% (127/160) of specimens that successfully clustered. Whole genome sequence assemblies were generated for five of the cyclosporiasis cases that were genotyped, including a hybrid assembly that improved the current reference assembly (GCF_002999335.1); this refined assembly was 44.2 Mbp in length (297 contigs, N50 value of 654019). These first molecular data generated from Canadian cyclosporiasis cases will support and inform future epidemiological investigations aimed at mitigating cyclosporiasis outbreaks.