Background: Candida species are opportunistic pathogens that can cause local or systemic invasive infections in patients hospitalized in intensive care units (ICUs). The present study aimed to rapidly and precisely identify Candida species, especially Candida auris, from patients hospitalized in high-risk units using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Materials and Methods: In this study, 136 clinical samples were prospectively collected from different organs of patients hospitalized in high-risk units recruited from three tertiary care training hospitals in Isfahan. Direct microscopic analysis using potassium hydroxide 10% followed by culturing on Sabouraud dextrose agar and CHROMagar culture media was primarily used to screen Candida species. Molecular detection of clinical isolates was mainly done using the PCR-RFLP method. Then, all the isolates were subjected to confirmatory diagnosis again through amplification of the C. auris-specific PCR. Results: Candida albicans was the most prevalent isolate (48. 8%), followed by Candida glabrata (17. 6%), Candida tropicalis (16. 1%), Candida parapsilosis (6. 6%), Candida krusei (5. 8%), Candida famata (4. 4%), Candida kefyr (2. 9%), and Candida guilliermondii (1. 4%). Therefore, the prevalence of non-albicans Candida species was higher than C. albicans isolates. No C. auris isolates were identified among 136 patients. However, according to the positive control, C. auris had no restriction enzyme cleavage site for MspI, and its PCR and digestion products were the same size (401 bp). Conclusions: The simple and reliable PCR-RFLP assay used in the study has the potential to identify rare yeasts such as C. auris. It can reduce turnaround times and costs if applied in developing countries.