The article presents a comprehensive study on the three-dimensional architecture of the yeast genome using a novel method that employs massively parallel sequencing to detect long-range chromosomal interactions. The method is capable of identifying interactions between DNA segments without prior knowledge of interacting sequences, and it has been validated using multiple controls and techniques. Key findings include the similarity in intra-chromosomal interaction density and the relationship between interaction frequency and spatial distance across different chromosomes. Chromosome XII exhibits a unique conformation due to the absence of interactions between its distal arms, suggesting the nucleolus as a barrier. Inter-chromosomal interactions are dominated by those between centromeres, with high interaction frequencies between telomeres of similarly sized chromosome arms. The findings support a Rab1 configuration where centromeres and telomeres occupy opposite poles of the nucleus. The study also reveals colocalizations of tRNAs, early replication origins, and chromosomal breakpoints, and highlights the flexibility of yeast chromosome arms compared to mammalian counterparts. A three-dimensional model of the yeast genome was constructed based on comprehensive mapping of chromosomal interactions. The method is compatible with next-generation sequencing technologies and has been validated through various controls, demonstrating its reliability and accuracy in detecting chromosomal interactions. The study provides insights into the structural organization of the yeast genome and has implications for understanding genome architecture in other organisms.The article presents a comprehensive study on the three-dimensional architecture of the yeast genome using a novel method that employs massively parallel sequencing to detect long-range chromosomal interactions. The method is capable of identifying interactions between DNA segments without prior knowledge of interacting sequences, and it has been validated using multiple controls and techniques. Key findings include the similarity in intra-chromosomal interaction density and the relationship between interaction frequency and spatial distance across different chromosomes. Chromosome XII exhibits a unique conformation due to the absence of interactions between its distal arms, suggesting the nucleolus as a barrier. Inter-chromosomal interactions are dominated by those between centromeres, with high interaction frequencies between telomeres of similarly sized chromosome arms. The findings support a Rab1 configuration where centromeres and telomeres occupy opposite poles of the nucleus. The study also reveals colocalizations of tRNAs, early replication origins, and chromosomal breakpoints, and highlights the flexibility of yeast chromosome arms compared to mammalian counterparts. A three-dimensional model of the yeast genome was constructed based on comprehensive mapping of chromosomal interactions. The method is compatible with next-generation sequencing technologies and has been validated through various controls, demonstrating its reliability and accuracy in detecting chromosomal interactions. The study provides insights into the structural organization of the yeast genome and has implications for understanding genome architecture in other organisms.