Grain polyphenol oxidase (PPO) activity can cause discoloration of wheat (Triticum aestivum L.) food products. Five crosses (PI 117635/Antelope; Fielder/NW03681; Fielder/Antelope; NW07OR1070/Antelope; NW07OR1066/OR2050272H) were selected to study the genetic inheritance of PPO activity. STS markers, PPO18, PPO29 and STS01, were used to identify lines with putative alleles at the Ppo-A1 and Ppo-D1 loci conditioning low or high PPO activity. ANOVA showed significant genotypic effects on PPO activity (P < 0.0001) in all populations. The generations and generation × genotype effects were not significant in any population. A putative third (null) genotype at Ppo-A1 (no PCR fragments for PPO18) was discovered in NW07OR1066 and NW07OR1070 derived populations, and these had the lowest mean PPO activities. Results demonstrated that both Ppo-A1 and Ppo-D1 loci affect the kernel PPO activity, but the Ppo-A1 has the major effect. In three populations, contrary results were observed to those predicted from previous work with Ppo-D1 alleles, suggesting the markers for Ppo-D1 allele might give erroneous results in some genetic backgrounds or lineages. Results suggest that selection for low or null alleles only at Ppo-A1 might allow development of low PPO wheat cultivars.