With the dramatic price dropping on next-generation sequencing, genotyping of a mapping or breeding population can be done in a short time at low cost directly by sequencing, termed as genotyping by sequencing (GBS). GBS is being developed and optimized at a good timing for the fast growing genetics and breeding methodologies, such as genome wide association study (GWAS) and genomic selection (GS). Alfalfa has a large genome (> 800 Mbp) and carries high level of heterozygosity. Therefore, to ensure the accuracy of genotyping by sequencing, the alfalfa whole genome needs to be reduced to increase sequencing depth. Restriction enzymes (REs) can be used to quickly, sufficiently and specifically reduce a genome. Cultivated alfalfa, Medicago Sativa ssp Sativa (L.), is autotetraploid, which poses another challenge -- allele dosage determination in an affordable and high throughput way. Deep coverage sequencing in GBS holds a hope for solving this issue. In order to apply GBS to alfalfa, we are currently trying to optimize a protocol developed for Maize and to figure out an optimal RE and the best multiplex level to obtain an ideal depth of sequencing by Illumina.