Abstract The YEASTRACT+ information system (http://YEASTRACT-PLUS.org/) is a wide-scope tool for the analysis and prediction of transcription regulatory associations at the gene and genomic levels in yeasts of biotechnological or human health relevance. YEASTRACT+ is a new portal that integrates the previously existing YEASTRACT (http://www.yeastract.com/) and PathoYeastract (http://pathoyeastract.org/) databases and introduces the NCYeastract (Non-Conventional Yeastract) database (http://ncyeastract.org/), focused on the so-called non-conventional yeasts. The information in the YEASTRACT database, focused on Saccharomyces cerevisiae, was updated. PathoYeastract was extended to include two additional pathogenic yeast species: Candida parapsilosis and Candida tropicalis. Furthermore, the NCYeastract database was created, including five biotechnologically relevant yeast species: Zygosaccharomyces baillii, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica and Komagataella phaffii. The YEASTRACT+ portal gathers 289 706 unique documented regulatory associations between transcription factors (TF) and target genes and 420 DNA binding sites, considering 247 TFs from 10 yeast species. YEASTRACT+ continues to make available tools for the prediction of the TFs involved in the regulation of gene/genomic expression. In this release, these tools were upgraded to enable predictions based on orthologous regulatory associations described for other yeast species, including two new tools for cross-species transcription regulation comparison, based on multi-species promoter and TF regulatory network analyses.