We developed a rapid genotypic assay to differentiate the germ tube-positive yeasts Candida albicans and Candida dubliniensis. Fluorescently labeled nucleic acid probe binding and subsequent denaturation from the target site in the PCR amplicons produced characteristic peak melting temperatures (T(m)) that identified each species. Peak T(m)s of C. albicans (n = 69) and C. dubliniensis (n = 28) isolates produced in the presence of their respective probes were 61.04 +/- 0.64 degrees C and 60.52 +/- 1.01 degrees C (averages +/- standard deviations). No signal was generated when the C. albicans or C. dubliniensis probes were tested against DNA from their counterparts. Both probes reacted with Candida tropicalis DNA, but the T(m) was 51.85 +/- 0.05 degrees C with the C. albicans probe and 51.92 +/- 0.10 degrees C with the C. dubliniensis probe, differentiating C. tropicalis DNA from C. albicans and C. dubliniensis. A novel hybrid probe was designed to identify both species in a single reaction based on a 4 degrees C difference in peak T(m)s. Our assay is rapid (</=2 h) and allows reliable detection and differentiation of the two germ tube-positive Candida spp.