Aim: To determine the structure of the chimeric chromosome X of bottom-fermenting yeasts. Methods and Results: Eight cosmid clones carrying DNA from chromosome X of bottom-fermenting yeasts were selected by end-sequencing. Four of the cosmid clones had Saccharomyces cerevisiae (SC)-type and Saccharomyces bayanus (SB)-type chimeric ends, two had SC-type ends and two had SB-type ends. Sequencing revealed that the bottom-fermenting yeast strains in this study had four types of chromosome X: SC-SC, SC-SB, SB-SC and SB-SB. The translocation site in the chimeric chromosome is conserved among bottom-fermenting yeast strains, and was created by homologous recombination within a region of high sequence identity between the SC-type sequence and the SB-type sequence. Conclusions: Existing bottom-fermenting yeast strains share a common ancestor in which the chimeric chromosome X was generated. Significance and Impact of the Study: The knowledge gained in this study sheds light on the evolution of bottom-fermenting yeasts.