Inheritance of trifluralin herbicide resistance was investigated for phenotype and genotype (molecular identification) in 12 segregating populations derived from crosses between resistant green foxtail (Setaria viridis) and foxtail millet cultivars (S. italica). Combining a herbicide bioassay and a bidirectional allele-specific polymerase chain reaction (PCR) assay, the trifluralin resistance in foxtail millet was proved to be conferred by a recessive α2-tubulin gene mutation at Ile-239, the allele transferred by green foxtail. However, a distorted segregation ratio in F2 populations was confirmed, with 16.9% resistant plants on average instead of the 25% expected for a single gene recessive mutation. This was not due to non-germinating seed and the distortion was also observed in the next generations, indicating a heritable phenomenon. After further crosses and self-fertilization, however, more advanced generations did not show the same pattern of skewed inheritance as in the early generations. These results suggest the possibility of linkage between the α2-tubulin gene and a modifier gene to explain the distorted segregation, which might be broken by crossing over.