An Axiom Sugarcane100K SNP array has been designed and successfully utilized to construct the sugarcane genetic map and to identify the QTLs associated with SCYLV resistance. To accelerate genetic studies in sugarcane, an Axiom Sugarcane100K single-nucleotide polymorphism (SNP) array was designed and customized in this study. Target enrichment sequencing 300 sugarcane accessions selected from the world collection of sugarcane and related grass species yielded more than four million SNPs, from which a total of 31,449 single-dose (SD) SNPs and 68,648 low-dosage (33,277 SD and 35,371 double dose) SNPs from two datasets, respectively, were selected and tiled on Affymetrix Axiom SNP array. Most of selected SNPs (91.77%) were located within genic regions (12,935 genes), with an average of 7.1 SNPs/gene according to sorghum gene models. This array was used to genotype 469 sugarcane clones, including one F1 population derived from the cross between Green German and IND81-146, one selfing population derived from CP80-1827, and 11 diverse sugarcane accessions as controls. Results of genotyping revealed a high polymorphic SNP rate (77.04%) among the 469 samples. Three linkage maps were constructed by using SD SNP markers, including a genetic map for Green German with 3482 SD SNP markers spanning 3336 cM, a map for IND81-146 with 1513 SD SNP markers spanning 2615 cM, and a map for CP80-1827 with 536 SD SNP markers spanning 3651 cM. Quantitative trait loci (QTL) analysis identified 18 QTLs controlling Sugarcane yellow leaf virus resistance segregating in the two mapping populations, harboring 27 disease-resistant genes. This study demonstrated the successful development and utilization of a SNP array as an efficient genetic tool for high-throughput genotyping in highly polyploid sugarcane.