To improve the hemagglutinin (HA) antigen yield of influenza A(H1N1)pdm09 candidate vaccine viruses, we generated 7:1, 6:2, and 5:3 genetic reassortant viruses between wild-type (H1N1)pdm09 (A/California/7/2009) (Cal7) and a high-yielding master virus, A/Puerto Rico/8/34 (PR8). These viruses contained the HA; HA and neuraminidase (NA); and HA, NA, and M genes, respectively, derived from Cal7, on a PR8 backbone. The influence of the amino acid residue at position 223 in Cal7 HA on virus growth and HA antigen yield differed between these reassortant viruses. NIIDRG-7, a 7:1 virus possessing arginine at position 223, exhibited a 10-fold higher 50% egg infectious dose (EID₅₀) (10.0 log₁₀EID₅₀/ml) than the 5:3 and 6:2 viruses. It also had 1.5- to 3-fold higher protein (13.8 μg/ml of allantoic fluids) and HA antigen (4.1 μg/ml of allantoic fluids) yields than the 5:3 and 6:2 viruses, which possessed identical Cal7 HA proteins. However, the HA antigen yield of the other 7:1 virus, which possessed glutamine at position 223 was 60% of that of NIIDRG-7. In addition, a novel 6:2 virus possessing Cal7 HA and the NA of A/Wisconsin/10/98 (a triple reassortant swine-like H1N1 virus), produced 107% of the HA yield of NIIDRG-7. In this study, we showed that the balance between HA and NA in the influenza A(H1N1)pdm09 virus affects its protein and antigen yield.