Few studies have targeted the relative performance of diagnostic methods used for the detection of Blastocystis, a unicellular organism often present in fecal specimens from individuals with and without gastrointestinal symptoms. Aims of this study included a comparison of the formol ethyl acetate concentration technique (FECT), permanent trichrome staining of feces fixed in sodium acetate-acetic acid-formalin (SAF-PST), xenic in vitro culture (XIVC), and polymerase chain reaction (PCR) regarding Blastocystis screening of 107 samples from 93 patients with suspected enteroparasitic disease. Compared with PCR, the sensitivity/specificity of XIVC, SAF-PST, and FECT was 89%/100%, 82%/100%, and 50%/100%, respectively. False-negative results generated by the FECT and SAF-PST appeared to be associated with Blastocystis sp. subtype 3. A comparison of results obtained by dideoxy sequencing of positive PCR products amplified from DNA extracted directly from feces and DNA extracted from 5- and 28-day-old XIVC of 10 randomly chosen Blastocystis isolates showed no disparities, indicating that XIVC has very little or no impact on subtype distribution or variation within a given specimen. It is recommended that short-term XIVC be used for cost-effective screening of fresh fecal specimens for Blastocystis infection to generate valid prevalence estimates and to identify isolates for molecular characterization in studies aiming to illuminate the molecular epidemiology of Blastocystis.