We developed a real-time LC PCR assay to detect a 152 bp sequence in an uncharacterized region of the Blastocystis genome. The described assay detected 11 of 11 ATCC strains of Blastocystis from subtypes 1, 3, and 4. Three of three stool samples from Oregon and California military personnel that were negative for Blastocystis by an ova and parasite test as well as a conventional PCR assay were positive for Blastocystis using our real-time LC PCR assay. Diagnosis of Blastocystis infections using this sensitive method, including DNA extraction and real-time PCR, only requires 3 h. The lower limit of detection for Blastocystis in stool using the real-time LC PCR assay was calculated to be 760 cells of Blastocystis per 100 mg of stool, an estimated 760 parasites per reaction. The assay did not cross-react with Ruminococcus hansenii, Anarococcus hydrogenalis, Bifidobacterium adolescentis, Fusobacterium prausnitzii, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, or Lactobacillus acidophilus. Because of the ease of use, sensitivity, specificity, and increase in Blastocystis infections in the USA we believe this assay has the potential to be useful as a clinical diagnosis tool of Blastocystis infection.