Context: Bloodstream infections (BSIs) caused by yeasts have an increasing frequency due to the growing population of immunosuppressed individuals. Among yeasts, Candida remains the most prevalent species with the increase in the incidence of non-albicans Candida species. Apart from Candida, other yeasts are also involved in causing BSI. High mortality associated with Candida and other yeast infection can be reduced by prompt and appropriate antifungal therapy. Hence, rapid identification and speciation of yeasts isolated from blood play a significant role in the management of the patients. Since conventional methods used for speciation of Candida and other yeasts are laborious, time-consuming and often unclear, rapid and accurate molecular techniques are required. Materials and Methods: Instead of using purified genomic DNA as template for polymerase chain reaction (PCR), we used yeast colony and cell suspensions in water and 0.10M potassium hydroxide as template for PCR. Candida albicans, Trichosporon and Cryptococcus neoformans were used as reference strains. Further, a total of 100 yeast isolates were also tested. All reactions were performed using the universal fungal primers ITS1 and ITS4; the PCR products were then digested with restriction enzyme (Msp1). Results: Direct colony PCR (DCPCR) produced sharp and distinct bands compared to the cell suspensions with the reference strains. All the 100 clinical isolates tested also produced distinct bands. Conclusion: DCPCR approach not only reduces the DNA template preparation time but is also easy, rapid and reduces the cost of PCR.