Candida species including species other than Candida albicans are of importance as causative agents of sepsis in intensive-care units, requiring prompt initiation of targeted therapy. While fluconazole is usually active against Candida albicans, non-Candida albicans species often require more sophisticated approaches. A rapid species diagnosis is therefore desirable and can be provided by fluorescence in situ hybridisation (FISH). However, broad evaluation studies of described probes are largely lacking and the probe panel that has been described is incomplete. As an addition to previously described C. albicans FISH probes, we evaluated published DNA probes for C. glabrata and C. krusei, as well as newly designed DNA probes for C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis, Crypotococcus neoformans and a group of intrinsically fluconazole-resistant Candida species for FISH with 22 reference strains, 23 well-characterised laboratory control strains, 169 isolates from clinical samples and 48 blood cultures. Sensitivity and specificity of >99% were demonstrated for all evaluated species-specific probes, whereas the probe that binds to a heterogeneous group of intrinsically fluconazole-resistant Candida species correctly identified eight of nine fluconazole-resistant clinical isolates. FISH yielded reliable results using the classical FISH procedure as well as a recently described slide chamber-based method. Given this good sensitivity and specificity, FISH may be applied for rapid identification of yeast in screening analyses, thus giving the opportunity for more precise targeting of antimycotic therapy.