Coccidiosis of domestic chicken is an important disease caused by any of seven species of Eimeria which, by developing within the epithelial cells of the intestine, cause lesions therein. We carried out a study on poultry farms located in various regions of Iran to determine the incidence and spread of Eimeria species by employing a single PCR test. A total of 64 fully confirmed clinically intestinal tracts were collected from different parts of Iran. From these 64 intestinal tracts, 82 samples were prepared from the different sites involved in the digestive tract. In morphological assessment, 23 samples could not be isolated and its information was not evaluated. Using morphological methods, the following seven species of Eimeria were identified: E. acervulina (15/59; 25.42%), E. tenella (30/59; 50.84%), E. maxima (12/59; 20.33%), E. praecox (1/59; 1.69%), E. necatrix (2/59; 3.38%), E. mitis (5/59; 8.47%), and E. mivati (2/59; 3.38%). Mixed infections were found in eight (13.55%) samples. In molecular assessment, 31 samples could not be isolated and its information was not evaluated. Totally, the following five species were identified using molecular methods: E. acervulina (35/51; 68.62%), E. tenella (33/51; 64.70%), E. maxima (6/51; 11.76%), E. brunetti (5/51; 9.80%), and E. necatrix (2/51; 3.92%). Mixed infections were found in 23 (45.09%) samples. The present study is an update on the situation of poultry coccidiosis in Iran and provides the first data on the molecular detection, identification, and characterization of Eimeria spp. in the poultry population of this country and confirmed the presence of different species of this parasite in this area. According to the results, E. acervulina and E. tenella, as the main disease-causing species, should be considered in control programs such as treatment and vaccination strategies.