Methods and compositions useful in the detection and identification of species of Candida are disclosed. The compositions are combinations of oligonucleotides, where the forward primers of the primer pairs have identical sequences, while each reverse primer of the primer pairs has a unique sequence relative to all of the other reverse primers; or the reverse primers of the primer pairs have identical sequences, while each forward primer of the primer pairs has a unique sequence relative to all of the other forward primers. The oligonucleotides also include probes capable of detecting these amplicons, and sequencing primers for determining, in primer extension reactions, the nucleotide sequences contained within the amplicons. The detection of an amplicon indicates that the sample contains at least one isolate of Candida albicans, Candida glabrata, Candida parapsilosis, or Candida tropicalis, and the nucleotide sequence data is used to determine which of these four Candida species is present.