Nosema disease is one of the most important diseases of adult honey bees worldwide. It is known as silent killer because there are no characteristic symptoms. The aim of the present study was to determine prevalence of Nosema species in various towns of Kurdistan province in Iran. A multiplex polymerase chain reaction (multiplex-PCR) was performed for identification of Nosema species infecting European honeybee, Apis mellifera. A total of 100 samples were collected from apiaries (870 hives) in 10 counties of Kurdistan province, located in the west of Iran. Samples were examined using light microscope and PCR. The light microscope was used to determine the presence of Nosema spores in all of the collected samples. Multiplex-PCR based on 16S ribosomal RNA was used to differentiate N. apis from N. ceranae. Overall prevalence of the microscopic evaluation and PCR method were 29.00% and 32.00%, respectively. The analysis of Nosema isolates from interrogation of DNA databank entries of Kurdistan apiaries (based on rRNA sequence data) indicated that only N. ceranae was widespread in these apiaries, and it had already been found in high percentages (50.00%) in Marivan and Kamiaran counties of Kurdistan province. It was shown that only N. ceranae was found by PCR assay in the region.