ABSTRACT An oligonucleotide microarray based on the arrayed-primer extension (APEX) technique has been developed to simultaneously identify pathogenic fungi frequently isolated from invasive and superficial infections. Species-specific oligonucleotide probes complementary to the internal transcribed spacer 1 and 2 (ITS1 and ITS2) region were designed for 24 species belonging to 10 genera, including Candida species ( Candida albicans , Candida dubliniensis , Candida famata , Candida glabrata , Candida tropicalis , Candida kefyr , Candida krusei , Candida guilliermondii , Candida lusitaniae , Candida metapsilosis , Candida orthopsilosis , Candida parapsilosis , and Candida pulcherrima ), Cryptococcus neoformans , Aspergillus species ( Aspergillus fumigatus and Aspergillus terreus ), Trichophyton species ( Trichophyton rubrum and Trichophyton tonsurans ), Trichosporon cutaneum , Epidermophyton floccosum , Fusarium solani , Microsporum canis , Penicillium marneffei , and Saccharomyces cerevisiae . The microarray was tested for its specificity with a panel of reference and blinded clinical isolates. The APEX technique was proven to be highly discriminative, leading to unequivocal identification of each species, including the highly related ones C. parapsilosis , C. orthopsilosis , and C. metapsilosis . Because of the satisfactory basic performance traits obtained, such as reproducibility, specificity, and unambiguous interpretation of the results, this new system represents a reliable method of potential use in clinical laboratories for parallel one-shot detection and identification of the most common pathogenic fungi.