Three ascomycetous yeast strains, H-6(T), ZX-15 and ZX-20, isolated from the bark of two tree species of the family Fagaceae collected from different regions of China, formed unconjugated and persistent asci containing two to four globose ascospores. 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region (including 5.8S rDNA) sequence analysis showed that they were closely related to the currently accepted Saccharomyces species with strong support. Comparisons of the rDNA sequences, electrophoretic karyotypes and physiological characters indicated that the three strains represent a novel species in the genus Saccharomyces. The name Saccharomyces arboricolus sp. nov. was proposed for the novel species, with H-6(T) (=AS 2.3317(T)=CBS 10644(T)) isolated from the bark of Quercus fabri as the type strain.