A multiplex-PCR Luminex xMAP bead probe fluid array using xTAG analyte-specific reagents (multiplex xTAG fungal ASR assay) was employed for detection of clinically significant Candida species, Cryptococcus neoformans, Histoplasma capsulatum, and Blastomyces dermatitidis from blood cultures. We tested 132 blood cultures negative (n = 10) or positive (n = 97) for yeasts and/or bacteria (n = 25). The assay showed sensitivity and specificity of 100% and 99%, respectively. The xTAG fungal ASR assay is a rapid assay that allows simultaneous identification of multiple yeast species.