Flocculation is important in beer fermentation because its affects both the process of fermentation and the flavor characteristics of the end product. In this study, meiotic segregant Saccharomyces cerevisiae Y7, which exhibits the NewFlo type flocculation desirable for brewing, was isolated from the top-fermenting yeast S. cerevisiae Y6. Next-generation sequencing showed that the Y7 genome contained two predicted genes (homologs of FLO5 and Lg-FLO1) that might be involved in NewFlo-type flocculation. Two transformants were constructed by inserting a drug resistance gene (kanMX6) into the respective genes. Flocculation was maintained when kanMX6 was inserted into the FLO5 homolog, but significantly reduced in the transformant when kanMX6 was inserted into one of the Lg-FLO1 loci. These results suggested that the Lg-FLO1 homolog is involved in the NewFlo-type flocculation of the top-fermenting yeast S. cerevisiae Y7. The bottom-fermenting S. pastorianus yeast strains commonly used in brewing are widely believed to derive from a natural hybrid of S. eubayanus and S. cerevisiae (i.e., a top-fermenting parent strain). Based on our results, it is speculated that the flocculation type and Lg-FLO1 gene of bottom-fermenting yeast are derived from top-fermenting yeast. These findings are expected to contribute to cross breeding work in the brewing industry and to the control of flocculation of top-fermenting yeast.