Modern sugarcanes are polyploid interspecific hybrids, combining high sugar content from Saccharum officinarumwith hardiness, disease resistance and ratooning of Saccharum spontaneum. Sequencing of a haploid S. spontaneum, AP85-441, facilitated the assembly of 32 pseudo-chromosomes comprising 8 homologous groups of 4 members each, bearing 35,525 genes with alleles defined. The reduction of basic chromosome number from 10 to 8 in S. spontaneumwas caused by fissions of 2 ancestral chromosomes followed by translocations to 4 chromosomes. Surprisingly, 80% of nucleotide binding site-encoding genes associated with disease resistance are located in 4 rearranged chromosomes and 51% of those in rearranged regions. Resequencing of 64 S. spontaneumgenomes identified balancing selection in rearranged regions, maintaining their diversity. Introgressed S. spontaneumchromosomes in modern sugarcanes are randomly distributed in AP85-441 genome, indicating random recombination among homologs in different S. spontaneumaccessions. The allele-defined Saccharumgenome offers new knowledge and resources to accelerate sugarcane improvement.