MIG-seq is one of the NGS library construction methods developed for ecological studies (Suyama and Matsuki, 2015). A library construction in this method can be started from low quality and/or small amount of DNA due to PCR at the initial step, but the number of polymorphisms that can be obtained is small, resulting in few applications for genetic analysis of crops. In this study, we hypothesized that MIG-seq could detect many polymorphisms in wheat, which has a relatively large genome, and we attempted to use MIG-seq for wheat genetic analysis. First, we investigated the relationship between genome size and the number of polymorphisms that can be obtained by MIG-seq using 12 plant species, and found that the larger the genome size, the more regions that can be sequenced by MIG-seq. Second, MIG-seq was applied to tetraploid wheat populations for population structure analysis and to the tetraploid wheat RILs and F2 population for linkage map construction and QTL analysis. We applied MIG-seq for 194 tetraploid wheat accessions and 8,207 SNPs/InDels with no defects were detected. Using these polymorphisms, the results of the PCA and phylogenetic tree construction and admixture analysis agreed with previous findings. Using genotype data of the RILs and F2 population obtained by MIG-seq, we could construct linkage maps with over 3,000 markers and QTLs for known flowering genes were detected. Finally, MIG-seq was also applied to the backcross lines to confirm the heterozygous regions of the backcross processes for NILs selection. When selecting NILs for the VRN-A3 locus using an emmer wheat accession TN26 as a donor parent of VRN-A3 and a durum wheat accession ‘Setodur’ as a recurrent parent, MIG-seq and CAPS marker were used to investigate the remaining heterozygous regions of each backcross generation. This resulted in the selection of individuals that may be heterozygous only at the VRN-A3 locus in the BC4F3 generation. These results indicate that MIG-seq is useful for high-throughput genotyping of wheat.