The lack of rapid diagnostic procedures is a major obstacle in the successful management of fungal disease. Most of the methods now used in the microbiology laboratory include growth-based phenotypic testing. However, Polymerase Chain Reaction (PCR) has allowed for rapid identification of organism without the need for a growing culture. An ion-paired reverse phase chromatography (IP-RP)-based assay using High performance liquid chromatography (HPLC) was developed with commercially available IP-RP-HPLC system called the WAVE to differentiate medically important fungi. Universal fungus-specific primers and Restriction Fragment Length Polymorphism (RFLP) of PCR amplicon were done prior to WAVE analysis. The assay was enhanced when combined with a searchable relational database of retention time. Discrimination among closely related fungal species was possible by evaluation of distinct-retention time patterns. This assay is simple, rapid, and allows for the identification of medically important fungi by searching the database with the information obtained from PCR-WAVE analysis.