Candida albicans is an opportunistic and prevalent human fungal pathogen causing the USA's fourth most hospital-acquired bloodstream infections. Few antifungal drugs are available to treat C. albicans, and some isolates of C. albicans has acquired resistance to these drugs. To develop the next generation of antifungal treatments, we must better understand what differentiates C. albicans from mammalian cells at the molecular level. This study focuses on the isoforms of pseudouridine synthase PUS1. PUS1A and PUS1B play crucial roles in RNA modification. Our proposed research seeks to understand the location of Pus1A and Pus1B proteins in yeast and filamentous forms of C. albicans upon heat stress. We designed a recyclable plasmid construct having GFP followed by an inducible Flippase and NatR gene (antibiotic resistance gene) flanked by the FRT site. Using this construct, we created C. albicans strains expressing either Pus1A-GFP or Pus1B-GFP. Using fluorescence microscopy, we demonstrated that, under normal growth temperature, Pus1A and Pus1B predominantly reside in the nucleus. However, upon heat shock, we observed a shift in localization of Pus1 isoforms from the nucleus to the cytoplasm. Our preliminary findings suggest a dynamic redistribution of these proteins, which may play a critical role in C. albicans response to heat shock and its pathogenicity. Future research should focus on understanding these proteins' molecular mechanisms and distinct roles in the context of different types of stress and C. albicans' pathogenicity.