Autotetraploid rice is a new germplasm resource obtained by doubling chromosomes through colchicine treatment. There have been many studies on the reproductive characteristics of autotetraploid rice, but little is known about global DNA variations and reasons for low embryo sac fertility in autotetraploid rice. Therefore, an autotetraploid rice line (T446) was used for resequencing and embryo sac fertility was observed. Whole-genome resequencing data revealed 87,229 SNPs and 11,022 InDels in the genome of T446 versus E246 (diploid rice), which had an average of 23.37 SNPs and 2.95 InDels per 100 kb. A total of 17,375 and 17,171 structural variations and 131 and 128 copy number variations were identified in the autotetraploid and its diploid counterpart, respectively. We detected 140 large-effects SNPs and InDel variants that might be related to the embryo sac fertility of autotetraploid rice, including 10 genes that may be closely associated with the development of the embryo sac. Of these, Os02g0292600 and Os06g0565200 were specifically expressed in the ovary. Mature embryo sac fertility was observed through whole-mount eosin B-staining confocal laser scanning microscopy. Many abnormalities were found in the embryo sac of T446, including embryo sac degeneration, embryo sac without female germ unit, abnormal polar nuclei, and poly-eggs, which, in turn, resulted in low seed set. However, whole-genome polymorphisms and genetic differences were high and exhibited broad prospects for genetic improvement. Genetic mutations in genes associated with embryo sac fertility in polyploid rice require further studies.