In this study, we aimed to investigate the incidence of Dientamoeba fragilis with different diagnostic methods in patients with gastrointestinal symptoms and determine the sensitivity and specificity of existing diagnostic methods. Fecal samples collected from 101 patients with gastrointestinal complaints (especially upper abdominal pain, abdominal and pelvic pain, nausea and vomiting, gastroenteritis and colitis, unexplained fever and diarrhea) and 20 control cases from various clinics were included in the study. Samples were first examined with native-Lugol (N-L) method and cultured in Robinson medium. All 121 stool and culture samples were stained with iron hematoxylin stain (IHS) and trichrome stain (TS) methods and examined by PCR and QPCR for D.fragilis. Among 121 stool samples 13 (10.7%), 2 (1.7%), 7 (5.7%) 13 (10.7%), and 7 (5.8%), 4 (3.3%), 2 (1.7%), 3 (2.5%) of cultured samples were determined positive with IHS, TS, PCR, QPCR respectively. Fifteen of the 121 stool samples were determined as diarrheal. All diarrheal stool samples were negative with IHS and TS. One of the diarrheal stools and 6 (4.9%) of the non-diarrheal stools were positive by PCR. All of the diarrheal stools were negative. Thirteen of the non-diarrheal stool samples (10.7%) were positive by QPCR. When the QPCR method was considered as gold standard, sensitivity and specificity values were determined as 46% and 93% in IHS, 0% and 99% in TS, 54% and 100% by PCR and sensitivity and specificity values were 67% and 96% in IHS, 33% and 98% in TS, 67% and 100% by PCR among cultured stool samples. As a result, it was determined that there was a statistically significant difference between the samples of the patients and the control groups and the sensitivity and specificity of the conventional and molecular methods (IHS, TS, PCR and QPCR) determined in this study supported the results of other compared studies. It has been determined that staining methods used for the diagnosis of D.fragilis gave false positivite or negativite results. In addition, the QPCR method is more advantageous in terms of time saving for the diagnosis and initiation of the treatment and in cases where QPCR is not available, IHS and conventional PCR methods should be used together. In our opinion, this study will contribute to the results of epidemiological and scientific studies on D.fragilis in Turkey.