Fifty-two yeast isolates from flowers and associated nitidulid beetles of the Brazilian Atlantic Forest (Mata Atlântica) region were found to represent a new species in the large-spored Metschnikowia clade. The species is heterothallic, haploid, and allogamous, and produces asci with two aciculate ascospores that can reach 80 μm in length, as is typical in the clade. Analysis of sequences of the ribosomal RNA gene cluster indicates that the new species is closely related to Metschnikowia lochheadii, which ranges across Central America to northern Brazil, occurs as an adventive species in Hawaii, but is rarely found in central Brazil. The species is not readily distinguishable from relatives based on morphology or growth responses, but is well delineated from M. lochheadii on reproductive isolation. Based on an intron splice site PCR screen, we selected 26 isolates for further study. The sequence of the region that includes the complete internal transcribed spacer/5.8S rRNA gene segment as well as the D1/D2 domains of the large subunit rRNA gene contained three polymorphic segments and 14 haplotypes were identified. Of these, a single divergent isolate from the southernmost of four sampled localities exhibited diminished mating success when crossed with others. We describe two varieties, Metschnikowia matae var. matae sp. nov. var. nov. (type UFMG-CM-Y395(T), CBS 13986(T), NRRL Y-63736(T); allotype UFMG-CM-Y391(A), CBS 13987(A), NRRL Y-63735(A)) and Metschnikowia matae var. maris sp. nov. var. nov. (type UFMG-CM-Y397(T), CBS 13985(T), NRRL Y-63737(T)). We also report on the discovery of the h (+) mating type of Candida ipomoeae and transfer of the species to Metschnikowia ipomoeae comb. nov. (allotype UWOPS 12-660.1(A), CBS 13988(A), NRRL Y-63738(A)).