Deoxyribonucleic acid (DNA) base composition and DNA base sequence relatedness comparisons were used for species delineation in the genus Kluyveromyces. Base composition values separated the members of the genus into three groups. The groups were further subdivided by comparing base sequences by using DNA/DNA renaturation experiments. Two DNA homology groups were identified. The first group included Kluyveromyces marxianus, Kluyveromyces fragilis, Kluyveromyces bulgaricus, Kluyveromyces cicerisporus, Kluyveromyces wikenii, and three anamorphs (Candida kefyr, Candida pseudotropicalis, and Torula cremoris); the members of this group exhibited .gtoreq. 90% DNA base sequence complementarity. The second group consisted of Kluyveromyces lactis, Kluyveromyces vanudenii, Kluyveromyces drosophilarum, and Kluyveromyces phaseolosporus; various pairs of these yeasts shared 64 to 98% of their DNA sequences. The two groups were only distantly related to each other (.ltoreq. 15% DNA base sequence complementarity). The other Kluyveromyces species appear to be unique, not being closely related to either of the two homology groups or to one another. Relationships deduced from comparisons of DNAs agreed well with those deduced by other workers from immunological comparisons of exo-.beta.-glucanases and from isoenzyme analysis but were only in partial agreement with a taxonomic arrangement made on the basis of mating studies. We propose recognition of the following species: Kluyveromyces aestuarii, Kluyveromyces africanus, Kluyveromyces blattae, Kluyveromyces delphensis, Kluyveromyces dobzhanskii, K. lactis (syn. K. drosophilarum, K. phaseolosporus, and K. vanudenii), Kluyveromyces lodderi, K. marxianus (syn. K. bulgaricus, K. cicerisporus, K. fragilis, and K. wikenii), Kluyveromyces phaffii, Kluyveromyces polysporus, Kluyveromyces thermotolerans, Kluyveromyces waltii, and Kluyveromyces wickerhamii.