CRISPR-Cas9-facilitated functional chromosome fusion allows the generation of a series of yeast strains with progressively reduced chromosome numbers that are valuable resources for the study of fundamental concepts in chromosome biology, including replication, recombination and segregation. We created a new yeast strain with a single chromosome by using the protocol for chromosome fusion described herein. To ensure the accuracy of chromosome fusions in yeast, the long redundant repetitive sequences near linear chromosomal ends are deleted, and the fusion orders are correspondingly determined. Possible influence on gene expression is minimized to retain gene functionality. This protocol provides experimentally derived guidelines for the generation of functional chromosome fusions in yeast, especially for the deletion of repetitive sequences, the determination of the fusion order and cleavage sites, and primary evaluation of the functionality of chromosome fusions. Beginning with design, one round of typical chromosome fusion and functional verifications can be accomplished within 18 d.