Population genetics studies of allopolyploid species lag behind those of diploid species because of practical difficulties in analysis of homeologs-duplicated gene copies originating from hybridized parental species. Pool-Seq, i.e. massive parallel sequencing of pooled individuals, has high potential for detecting nucleotide polymorphisms within and among multiple populations; however, its use has been limited to diploid species. We applied Pool-Seq to an allopolyploid species by developing a bioinformatic pipeline that assigns reads to each homeolog as well as to each polymorphic allele within each homeolog. We simultaneously sequenced eight genes from twenty individuals from each of 24 populations, and found over 100 polymorphic sites in each homeolog. For two sites, we estimated allele frequencies using the number of reads and then validated these estimations by making individual-based estimations. Pool-Seq using our bioinformatic pipeline allows efficient evaluation of nucleotide polymorphisms in a large number of individuals, even in allopolyploid species.