Non–albicans Candida (NAC) is known as the most common cause for the opportunistic infections.. In the present study we identify Candida species isolated from various clinical specimens by using phenotypic and genotypic method. A total of 100 samples were examined by conventional methods and the genotypic analysis was done by using polymerase chain reaction– restriction fragment length polymorphism (PCR–RFLP) technique. The internal spacer region (ITS) of the fungal rRNA genes was recruited for PCR amplification of target sequences and MspI enzyme was used to digest PCR amplicons. By using genotypic method, we successfully identified all clinical isolates of Candida. Among them C.tropicalis was identified the most common species followed by C.albicans, C.parapsilosis, C.glabrata, C.krusei, C.guilliermondii. PCR–RFLP using ITS1 and ITS4 primers and restriction enzyme MspI is a simple, rapid, and cost–effective method for the differentiation between Candida species that is applicable in clinical laboratories.