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The increasing demand for craft beer is driving the search for novel ale yeast cultures from brewing-related wild environments. The focus of bioprospecting for craft cultures is to identify feral yeasts suitable to imprint unique sensorial attributes onto the final product. Here, we integrated phylogenetic, genotypic, genetic, and metabolomic techniques to demonstrate that sour beer during aging in wooden barrels is a source of suitable craft ale yeast candidates. In contrast to the traditional lambic beer maturation phase, during the aging of sour-matured production-style beer, different biotypes of <i>Saccharomyces cerevisiae</i> dominated the cultivable in-house mycobiota, which were followed by <i>Pichia membranifaciens</i>, <i>Brettanomyces bruxellensis</i>, and <i>Brettanomyces anomalus</i>. In addition, three putative <i>S. cerevisiae</i> × <i>Saccharomyces uvarum</i> hybrids were identified. <i>S. cerevisiae</i> feral strains sporulated, produced viable monosporic progenies, and had the <i>STA1</i> gene downstream as a full-length promoter. During hopped wort fermentation, four <i>S. cerevisiae</i> strains and the <i>S. cerevisiae</i> × <i>S. uvarum</i> hybrid WY213 exceeded non-<i>Saccharomyces</i> strains in fermentative rate and ethanol production except for <i>P. membranifaciens</i> WY122. This strain consumed maltose after a long lag phase, in contrast to the phenotypic profile described for the species. According to the <i>STA1</i>+ genotype, <i>S. cerevisiae</i> partially consumed dextrin. Among the volatile organic compounds (VOCs) produced by <i>S. cerevisiae</i> and the <i>S. cerevisiae</i> × <i>S. uvarum</i> hybrid, phenylethyl alcohol, which has a fruit-like aroma, was the most prevalent. In conclusion, the strains characterized here have relevant brewing properties and are exploitable as indigenous craft beer starters.