Real-time polymerase chain reaction (PCR) is currently considered the most sensitive method to detect low abundance DNA of pathogens in clinical samples. Furthermore, obtaining DNA sequence is the 'gold standard' of precise molecular detection. Here we combine species-specific real-time PCR and pyrosequencing to rapidly amplify and sequence ribosomal DNA from Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis, which are commonly associated with candida vaginitis (CV). A standard curve was developed from plasmids containing the target DNA for each of the Candida species. A minimum real-time PCR and pyrosequencing detection limit of 100 copies per reaction was achieved. The combined technique was applied to the identification of the four Candida species in DNA extracts from vaginal samples. The results from 231 samples were compared with conventional PCR methods of identification. The results of both methods agreed on all but two samples, which were determined by both methods to contain C. albicans, but real-time PCR and pyrosequencing identified a second species that went undetected by conventional PCR. This is the first application of real-time PCR and pyrosequencing to DNA from vaginal samples for identification of four Candida species associated with CV, without the need for time-consuming culture methods.