The diagnosis of candidemia is important for prompt initiation of antifungal therapy. Two hundred twenty-five patients at high risk for candidemia who had blood cultures drawn and were hospitalized for more than 15 days were followed-up prospectively over a 2-year period. Polymerase chain reaction (PCR) and whole-blood cultures monitored by the automated BactAlert system (Organon Teknika, Durham, NC, USA) were used to detect candidemia in all patients hospitalized in high-risk areas for more than 15 days. DNA was extracted and amplified using ITS5 and ITS4 base pair primers, and the PCR products were sequenced for identification of Candida spp. A blood culture positive for Candida was considered the gold standard for diagnosis of candidemia. Variables associated with the development of candidemia diagnosed by positive blood culture were also evaluated in the patients. The overall mortality rate was 26.1%. Mortality in candidemic patients was 41.9% and in noncandidemic patients 22.5% (p = 0.009). PCR sensitivity and specificity were 72.1 and 91.2%, respectively. Positive and negative predictive values were 65.9 and 93.2%, respectively. The logistic regression of the multivariate analysis showed that parenteral nutrition (p < 0.0001), fever (p = 0.01), neutropenia (p = 0.04), and an indwelling urinary catheter (p = 0.02) were significant variables associated with the development of candidemia. The PCR technique in conjunction with DNA sequencing was a helpful tool in the diagnosis of candidemia.