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Fourteen apiculate yeast strains isolated from various sources in South Africa, North America and the Hawaiian islands were found to be genetically divergent from other Hanseniaspora-Kloeckera species by using randomly amplified polymorphic DNA (RAPD)-PCR. After cluster analysis of the RAPD-PCR fingerprints, five groups were recognized. DNA reassociation values among representatives of these groups and strains of Hanseniaspora-Kloeckera species revealed that the strains represent five novel species. Four are described here as novel species of HANSENIASPORA: Hanseniaspora meyeri sp. nov. (type CBS 8734(T)), Hanseniaspora clermontiae sp. nov. (type CBS 8821(T)), Hanseniaspora lachancei sp. nov. (type CBS 8818(T)) and Hanseniaspora opuntiae sp. nov. (type CBS 8733(T)). The fifth novel species, which is represented by only a single strain, CBS 8772, is not introduced as a new taxon. Phylogenetic analyses of the D1/D2 region of the 26S rDNA and internal transcribed spacer (ITS) regions with 5.8S rDNA sequences placed H. meyeri, H. clermontiae, H. lachancei, H. opuntiae and strain CBS 8772 close to Hanseniaspora uvarum and Hanseniaspora guilliermondii. The key characteristics for standard physiological identification of H. clermontiae and H. lachancei were respectively maximal growth temperature and assimilation of 2-keto-D-gluconate. However, physiological characteristics did not allow the distinction of H. opuntiae and strain CBS 8772 from H. guilliermondii or H. meyeri from H. uvarum. These three novel taxa can be identified by either ITS sequencing or PCR-RFLP of ITS regions using restriction enzymes MboII and HinfI.