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Although fermented foods are an important part of the human diet and have seen an increasing interest nowadays, some challenges still need to be solved. Specifically, yeast identification through culture-independent methodologies is still limited to the genus level. Unlike bacterial species identifications, long-read sequencing technologies have barely been used for yeast species identification, and, to the best of the authors\u2019 knowledge, it has not been validated with mock communities reflecting food fermentation processes yet. Therefore, in the current study, we present an amplicon-based metabarcoding approach targeting the full-length internal transcribed spacer (ITS) region comprising ITS1, the 5.8S rRNA gene, and ITS2, using the PacBio HiFi sequencing platform. This method was validated using DNA-based mock communities composed of yeast species involved in sourdough, lambic beer, and cocoa fermentation processes. Accurate species-level identification was achieved for most of the species. However, special attention should be given to\n                      <jats:italic toggle=\"yes\">Saccharomyces<\/jats:italic>\n                      -rich niches, as accurate species-level identification for this genus is still challenging. Furthermore, underestimation of the relative abundance of species with short ITS regions, such as\n                      <jats:italic toggle=\"yes\">Pichia<\/jats:italic>\n                      and\n                      <jats:italic toggle=\"yes\">Brettanomyces<\/jats:italic>\n                      , occurred. In addition, the method was successfully applied to describe the yeast diversity present in two sourdough and two lambic beer samples. Overall, the current method provides an unprecedented way of determining the species-level yeast composition of complex ecosystems present in fermented food products.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>To date, species-level identification of common yeasts present in food fermentation ecosystems has been difficult, if not impossible, when using short-read sequencing methods. However, species-level identification is essential when evaluating and describing the characteristics of fermented food microbiomes. The current study reports on the development and validation of an amplicon-based metabarcoding approach combined with long-read PacBio HiFi sequencing targeting the full internal transcribed spacer (ITS) region, comprising the ITS1 and ITS2 regions, as well as the 5.8S rRNA gene. The described methodology enables species-level identification of the most common yeasts present in food fermentation ecosystems. This new methodology provides an important tool not only for the investigation of fermented foods but also for other fields engaged in complex microbial community analysis.<\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/spectrum.00060-26","type":"journal-article","created":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T13:04:14Z","timestamp":1783343054000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":1,"title":["Unraveling yeast diversity in food fermentation using ITS1-2 amplicon-based metabarcoding"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4949-262X","authenticated-orcid":true,"given":"Ines","family":"Pradal","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/006e5kg04","id-type":"ROR","asserted-by":"publisher"}],"name":"Research Group of Industrial Microbiology and Food Biotechnology (IMDO), Faculty of 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