The genome is packed into the nucleus in a highly organized manner, and this organization has consequences for transcriptional regulation, DNA break repair, and other nuclear processes. In Saccharomyces budding yeasts, the genome is positioned in a Rabl orientation throughout the cell cycle, with centromeres attached to the spindle pole, the nucleolus opposite to the spindle pole, and telomeres at the nuclear periphery. Although various studies have suggested additional features of Saccharomyces budding yeast nuclear organization, such as clustering of tRNA genes and origins of replication, genome-wide chromatin conformation capture (Hi-C) data from haploids lack evidence of long-range interactions driven by DNA sequence and can largely be explained by simple polymer models simulating a Rabl configuration. Further, although FISH- and recombination-based studies have suggested mitotic pairing of homologous chromosomes in diploid yeast, whether mitotic pairing occurs remains controversial and has not been interrogated in a genome-wide manner. To systematically uncover the 3D organization of the diploid budding yeast genome, we performed Hi-C on interspecific hybrids of S. cerevisiae, S. paradoxus, and S. uvarum during exponential growth and stationary phase. Here, we report that the organization of diploid Saccharomyces genomes, unlike those of haploids, substantially deviates from the simple polymer model. First, the homologous chromosomes carrying the rDNA arrays interact preferentially, even at their centromeres. Although preferential pairing of sequences near the rDNA arrays could be predicted by simple polymer models, the pairing of the centromeres is less easily explained. Second and even more surprisingly, we find that specific homologous sequences on chromosome 13 interact with each other, only in stationary phase. A deletion of one copy of this region in S. cerevisiae x S. uvarum eliminated the interaction, and sequentially smaller deletions allowed us to narrow down the source of the interaction to a ~1 kb intergenic region. Unlike previously reported interactions between genes relocated to the nuclear pore complex upon induction, this novel interchromosomal interaction appears to be repressive, as the region is bound by stress-induced transcriptional repressor Xbp1 and is flanked by genes repressed during stationary phase. Finally, we show that in both hybrids of diverged S. cerevisiae strains and S. cerevisiae x S. paradoxus hybrids, interactions between homologous regions are subtly but clearly enriched even after controlling for their distances from centromeres; we conclude that in diploid budding yeasts, mitotic pairing of homologous chromosomes occurs genome-wide.