Microsporidia, obligate intracellular parasites, were first defined by Nageli in 1857. Microsporidia phylum consists of 200 genus and 1500 species. They have a wide host spectrum including insects, fish, and mammals. It has been shown that they may also infect humans and may be existed both in symptomatic and asymptomatic forms. There are eight species infecting humans, which include Anncaliia (Brachiola, Nosema), Encephalitozoon, Entrocytozoon, Microsporidium, Nosema, Pleistophora, Trachipleistophor, and Vittaforma. The species most commonly infect humans are Encephalitozoon intestinalis and Enterocytozoon bieneusi. The aim of this study was to determine the prevalence of Microsporidia by using two different chemiluminescence stains, namely uvitex 2B and calcoflour and detect species by molecular analysis in diarrheal patients. For this purpose, we studied stool samples of 200 patients with diarrhea sent to Gazi University Health Practice and Research Hospital, Microbiology Laboratory and Ankara Numune Training and Research Hospital Microbiology Laboratory between 2012-2013. The stool samples were stained with chemiluminescent stains uvitex 2B and calcoflour methods; the Microsporidia prevalence was found to be 38% (77/200) by fluorescent microscopic examination. Statistically an excellent consistency was found between the chemiluminescent stains uvitex 2B and calcoflour (Cohen's kappa=0.881). A statistical analysis for the consistency of uvitex 2B and calcoflour in terms of numerical density (low, high) and luminescence of spores (dim, bright) showed a moderate consistency between the two stains with respect to determining numerical density of spores (Cohen's kappa=0.354), while there was no consistency in terms of luminescence of spores (Cohen's Kappa=0.001). No significant difference was found between the Microsporidia prevalence with respect to age group or clinics (p > 0.05). A sex-based analysis showed that Microsporidia prevalence was more common in women (50%) than men (30.8%) (p < 0.05). In 77 samples that were detected positive for Microsporidia with uvitex 2B and calcoflour stains determination of genus and species level were done by using multiplex nested polymerase chain reaction (PCR) method. With this technique, seven (9.1%) of 77 isolates were detected as E.bieneusi, and 70 (90.9%) as Encephalitozoon spp. When the Microsporidia genus was considered, the Microsporidia prevalence did not show differences with respect to age, sex, and referring clinics (p > 0.05). In our study 44 (62.9%) of 70 Encephalitozoon spp. were E.intestinalis, 22 (31.4%) were E.cuniculi, and 4 (5.7%) were E. hellem. No statistical difference was found in the distribution of Encephalitozoon spp. with age, sex, and referring clinic (p > 0.05). We concluded that examination of stool samples with the chemiluminescent stain uvitex 2B and/or calcoflour would be useful for the initial stage of Microsporidia diagnosis; furthermore, the multiplex nested PCR method was considered useful for determination of genus and species. In our country, there is a small number of molecular reports about Microsporidia prevalence in stool samples. Molecular methods should be used more commonly for the evaluation of treatment options in diarrheal patients and detection of Microsporidia epidemiology.