We have previously developed a chromosome-splitting technique based on homologous recombination in Saccharomyces cerevisiae. To facilitate chromosome splitting at multiple sites, we focused on the delta sequences that are distributed in more than 200 copies throughout the yeast genome. We constructed a new chromosome-splitting vector harboring the YFLW delta4 sequence and the hisG-URA3-hisG cassette, and transformed yeast cells with this vector. The karyotype analysis of transformants showed that chromosomes XIV, III, and IV, or other chromosomes are split. After the excision of the URA3 gene, the transformant with split chromosome IV was subsequently transformed with the same vector. Karyotype analysis revealed that further splitting occurred at chromosome X, the split chromosome IV, or other chromosomes. These results indicate that delta sequences are efficient target sites for repeated chromosome splitting at multiple sites with a single vector.