Honey is widely produced in China as a natural health food with high nutritional value. In this study, high-throughput sequencing (HTS) and culture-dependent methods were used to assess the yeast diversity of wild honey from three different regions of Xinjiang. Analysis of HTS data of the 26S rRNA gene NL1F and NL2R regionsrevealed the highest number of operational taxonomic units (OTUs) of the yeast genus Zygosaccharomyces in honey, followed by the genera Aureobasidium and Clavispora. Multifloral honeynot only differed in yeast structure and composition from monofloral honey but also demonstrated greater diversity. Thirty-five yeast strains were obtained by traditional isolation and culture methods, and their morphological, physiological, biochemical, and 26S rRNA D1/D2 domain genes were sequenced to identify 11 yeast species: Zygosaccharomyces siamensis, Aureobasidium pullulans, Naganishia albida, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Rhodotorula glutinis, Pichia kluyveri, Barnettozyma californica, Rhodotorula kratochvilovae, Candidatropicalis, and Pichia terricola. The species identified using this approach differed somewhat from those revealed by high-throughput sequencing. The results of this study provide basic information on the yeast diversity of native honey in Xinjiang, which will help to develop germplasm resources of yeast in honey.