Reactive oxygen species (ROS) play important roles during anther and pollen development. DNA damage may cause chromosome fragmentation that is considered to underlie chromosome elimination for haploid induction by matrilineal pollen, a key step in MATRILINEAL-based double haploid breeding technology. But when and how DNA damage occurs is unknown. We performed comparative studies of wheat pollens from the wild-type and the CRISPR/Cas9 edited matrilineal mutant (mMTL). Chemical assays detected a second wave of ROS in mMTL pollen at the three-nuclei-stage and subsequently, along with reduced antioxidant enzyme activities. RNA-seq analysis revealed disturbed expression of genes for fatty acid biosynthesis and ROS homoeostasis. Gas chromatography-mass spectrometry measurement identified abnormal fatty acid metabolism that may contribute to defective mMTL pollen walls as observed using electron microscopy, consistent with the function of MTL as a phospholipase. Moreover, DNA damage was identified using TdT-mediated dUTP nick-end labelling and quantified using comet assays. Velocity patterns showed that ROS increments preceded that of DNA damage over the course of pollen maturation. Our work hypothesises that mMTL-triggered later-stage-specific ROS causes DNA damage that may contribute to chromosome fragmentation and hence chromosome elimination during haploid induction. These findings may provide more ways to accelerate double haploid-based plant breeding.