To elucidate the effects of different glutenin subunit alleles on the parameters of wheat flour quality, we analyzed the dough and gluten properties using a doubled-haploid population derived from a cross between two wheat cultivars differing in the glutenin subunit loci except for Glu-D3, namely Grandin (Glu-A1b, Glu-B1c, Glu-D1d, Glu-A3f, Glu-B3h and Glu-D3a) and Kitamiharu 57 (Glu-A1a, Glu-B1i, Glu-D1a, Glu-A3c, GluB3b and Glu-D3a). Based on the analysis of the DH lines, no significant differences in the flour protein content were detected among the glutenin alleles. However, the Glu-D1 and Glu-B3 alleles exerted significant effects on the dough and gluten properties. The DH lines with the Glu-D1d or Glu-B3b alleles showed a significantly higher gluten index, determined using the Glutomatic system and longer dough development time determined using a mixograph. The Glu-D1d and Glu-B3b alleles additively increased the dough and gluten strength. The DH lines with the Glu-A1a allele also showed a significantly higher gluten index and longer dough development time than those with the Glu-A1b allele, though to a lesser extent. These results indicated that the Glu-B3b allele exerted similar effects on the dough and gluten strength as the Glu-D1d allele. Since the DH lines harboring the Glu-D1d and Glu-B3b alleles showed excessive values for the strength of dough and gluten, it was suggested that both the Glu-1 and Glu-3 allelic composition should be considered to improve the wheat flour quality.