Due to recent outbreaks of cyclosporiasis associated with consumption of fresh berries, producers are demanding modern microbiological tools for the rapid and accurate identification of the human pathogen in berries and environmental samples. The aim of the present work was to develop a molecular tool based on a PCR approach for the rapid and accurate detection of . A nested PCR assay was validated for the amplification of a 294 bp size region of the gene from . The limit of detection for the nested PCR assay was validated using 48 berry samples spiked with ~0, 10, 100, and 1000 oocyst per gram of sample. With this assay, it was possible to detect as few as 1 oocyst per gram of berry, in a 50 g sample. Sanger DNA sequencing and phylogenetic analysis were carried out to confirm the presence of in berry (n = 17) and soil (n = 5) samples. The phylogenetic analysis revealed that the sequences obtained from Mexico clustered within a group recovered from China, Peru, Guatemala-Haiti, and Japan. The PCR protocol designed in the present study could be an important tool for the rapid and accurate detection of this human pathogen in environmental and food samples.