Aspergillus species (sp.) that causes opportunistic infections have been increasingly found in human mainly immunosuppressive patients around the world every year. The main objective was to use a rapid and cheap molecular method for monitoring Aspergillus infections and epidemiological approaches. In order to identity Aspergilli species (spp.), a number of molecular methods including restriction fragment length polymorphism (RFLP) have been employed in accordance with ribosomal RNA amplification. The focus of this study — a group of hospitalized patients with clinical and subclinical signs of infection. All of the collected clinical specimens were transported to the medical mycology lab and examined for Aspergillus identification. The environmental specimens were collected from air and surfaces inspected for the Aspergillus within the hospital sources. At first, growth characteristics and microscopic features on mycological media for the identification of Aspergillus sp. were performed. For the confirmation of Aspergillus isolates which similarly found in clinical and environmental sources, molecular method polymerase chain reaction/restriction fragment length polymorphism was carried out. From the mentioned specimens, 102 fungal isolates included Candida spp., Aspergillus spp. and other fungi. Aspergillus flavus (47%), Aspergillus fumigatus (29.4%) and Aspergillus niger (23.5%) all were found as the most common clinical isolates. In addition, Aspergillus isolates from environmental were Aspergillus niger (43.7%), Aspergillus flavus (41.7%), Aspergillus fumigatus (14.6%). Therefore, polymerase chain reaction-restriction fragment length polymorphism with a single restriction enzyme can be very useful in the identification of Aspergillus spp., because of its facility in use, speed, robust, and high sensitivity of diagnosis.