The multidrug-resistant opportunistic yeast species of Candida auris, Candida haemulonii, Candida duobushaemulonii and Candida pseudohaemulonii continue to endanger the healthcare settings around the globe. Due to the lack of a specific qPCR assay for detection of these species from clinical samples, we developed a multiplex qPCR assay. Analytical specificity and sensitivity showed 100% specificity and the sensitivity of up to ten genomes of target species with a high value of reproducibility (R2 >0.99). Subsequently, from spiked serum samples, our qPCR specifically could detect up to ten genomes of C. auris and one genome of C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii (R2 >0.98). Lack of cross reaction with the human DNA, a high degree of specificity and sensitivity, showed the potential of our multiplex PCR for direct detection of C. auris and closely related species from serum samples of suspected patients. Future studies are warranted to assure its applicability in clinical settings.