Aegiolops kotschyi cytoplasmic male sterile system often results in part of haploid plants in wheat (Triticum aestivum L.). To elucidate the origin of haploid, 235 wheat microsatellite (SSR) primers were randomly selected and screened for polymorphism between haploid (2n = 3x = 21 ABD) and its parents, male-sterile line YM21 (2n = 6x = 42 AABBDD) and male fertile restorer YM2 (2n = 6x = 42 AABBDD). About 200 SSR markers yielded clear bands from denatured PAGE, of which 180 markers have identifiable amplification patterns, and 20 markers (around 8%) resulted in different amplification products between the haploid and the restorer, YM2. There were no SSR markers that were found to be distinguishable between the haploid and the male sterile line YM21. In addition, different distribution of HMW-GS between endosperm and seedlings from the same seeds further confirmed that the haploid genomes were inherited from the maternal parent. After haploidization, 1.7% and 0.91% of total sites were up- and down-regulated exceeding twofold in the shoot and the root of haploid, respectively, and most of the differentially expressed loci were up/down-regulated about twofold. Out of the sensitive loci in haploid, 94 loci in the shoot, 72 loci in the root can be classified into three functional subdivisions: biological process, cellular component and molecular function, respectively.