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Background: Reports on microsporidia infections are increasing and some species, such as Enterocytozoon bieneusi and Encephalitozoon intestinalis, have become important causes of chronic malabsorptive diarrhea, especially in HIV infected patients. In this study, Modified Trichrome-Blue (MTS) and Acid-Fast Trichrome (AFT) staining methods as well as PCR technique were used for detecting microsporidia in stool specimens. Materials and Methods: In this experimental study, a total of 71 stool specimens from AIDS patients with chronic diarrhea were collected and sent to laboratory. Two slides were prepared for each stool specimen. The slides were fixed with methanol, stained with MTS and AFT stain, and read by at least three individuals. In addition, PCR with primers directed to a conserved region of the 16s rRNA of intestinal microsporidian spores was used. Results: Totally of 71 patients, 13 patients (18.30%) were positive for microsporidia by MTS and AFT stain methods. In addition, 9 patients (12.67%) were positive for cryptosporidium by AFT stain and 4 (5.63%) of them were positive for microsporidia. Furthermore, 16 patients (22.53%) were positive for intestinal microsporidiosis by PCR technique. Notably, all cases that were positive for microsporidia by staining methods were also positive for PCR technique as well Conclusion: PCR technique was more sensitive than staining methods. Also, MTS and AFT stain methods were equally useful in the diagnosis of microsporidiosis.