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<i>Cyclospora cayetanensis</i> is a coccidian parasite that causes diarrheal illness outbreaks worldwide. The development of new laboratory methods for detection of <i>C. cayetanensis</i> is of critical importance because of the high potential for environmental samples to be contaminated with a myriad of microorganisms, adversely impacting the specificity when testing samples from various sources using a single molecular assay. In this study, a new sequencing-based method was designed targeting a specific fragment of <i>C. cayetanensis</i> <i>cytochrome oxidase gene</i> and developed as a complementary method to the TaqMan qPCR present in the U.S. FDA BAM Chapter 19b and Chapter 19c. The comparative results between the new PCR protocol and the qPCR for detection of <i>C. cayetanensis</i> in food and water samples provided similar results in both matrices with the same seeding level. The target region and primers in the protocol discussed in this study contain sufficient <i>Cyclospora</i>-specific sequence fidelity as observed by sequence comparison with other Eimeriidae species. The sequence of the PCR product appears to represent a robust target for identifying <i>C. cayetanensis</i> on samples from different sources. Such a sensitive method for detection of <i>C. cayetanensis</i> would add to the target repertoire of qPCR-based screening strategies for food and water samples.