The chitin synthase structural gene, WdCHS5 was isolated from Wangiella dermatitidis. Sequence analysis revealed that this gene has a myosin motor-like-encoding region at its 5′-end and a chitin synthase (class V)-encoding region at its 3′-end. The sequence analysis also revealed that the 5,655-bp open reading frame of WdCHS5 encoded 1,885 amino acids, and had a 53-bp intron near one end and a 57-bp intron near the other. Northern blotting showed that WdCHS5 was highly expressed under conditions of stress, such as temperature shift from 25°C to 37°C, or conditions that induce morphology changes. Analysis of the 5′ upstream region of WdCHS5 indicated that one or more of the potential regulatory elements detected might have contributed to the high expression. Disruption of WdCHS5 by different methods produced mutant strains with temperature-sensitive phenotypes, which grew normally at 25°C, but had severe growth and cellular abnormalities at 37°C. The observation that supplementing osmotic stabilizers, such as sorbitol and sucrose could rescue the phenotype indicated that the loss of WdChs5p caused the cell wall integrity defects. Animal survival tests using a mouse model of acute-infection showed that all wdchs5∆ strains were less virulent than the wild-type parental strain. Fungal organ burden assays demonstrated that the wdchs5∆11 mutant could not survive long enough in mice to cause any infection. Reintroduction of the WdCHS5 gene into wdchs5∆ mutants rescued the ts-phenotype and reestablished their virulence. Four double chitin synthase gene disruption mutants involving WdCHS5 were constructed. Three of them showed similar phenotype as that of the wdchs5∆ mutant. However, the double disruption of WdCHS4 and WdCHS5 resulted in a mutant which had the combined phenotype of WdCHS4 and WdCHS5 single gene disruption mutants and grew very poorly in a less rich medium. This report documents that the product of WdCHS5 is required for the sustained growth of W. dermatitidis at 37°C, and this is of critical importance to its virulence.