Puroindoline alleles, grain characteristics, milling performances and physical properties of flour of 22 Korean wheat cultivars were evaluated to determine the influence of puroindolines genotypes on grain and flour characteristics and to provide useful information for improving milling and end-use quality in Korean wheat breeding programs. Nine Korean wheat cultivars carried with Pina-D1a/Pinb-D1a, 11 cultivars had Pina-D1a/Pinb-D1b and 2 cultivars were Pina-D1b/Pinb-D1a. Korean wheats carrying with Pina-D1a/Pinb-D1a genotypes showed lower test weight and thousand kernel weight, area and roundness of grain and hardness index in grain characteristics, lower flour yield and higher proportion of break flour in milling performances and lower average particle size of flour, ash and damaged starch content, water retention capacity, yellowness-blueness and higher lightness of flour than wheats with Pina-D1a/Pinb-D1b or Pina-D1b/Pina-D1a genotypes. Pina-D1a/Pinb-D1b genotypes showed lower 1000-kernel weight, grain area, higher average of particle size of flour, higher ash and damaged starch content than Pina-D1b/Pina-D1a genotypes. There was no difference in hardness index of grain, milling performances, flour color between Pina-D1a/Pinb-D1b and Pina-D1b/Pina-D1a genotypes. These results could present the information to improve milling quality and physical properties of flour in Korean wheat breeding programs.