Autotetraploid rice is a useful germplasm for polyploid rice breeding, however, low seed setting is the major barrier in commercial utilization of autotetraploid rice. Our research group has developed neo-tetraploid rice lines, which have the characteristics of high fertility and heterosis when crossed with autotetraploid rice. In the present study, re-sequencing and RNA-seq were employed to detect global DNA variations and differentially expressed genes (DEGs) during meiosis stage in three neo-tetraploid rice lines compared to their parents, respectively. Here, a total of 4109881 SNPs and 640592 InDels were detected in neo-tetraploid lines compared to the reference genome, and 1805 specific presence/absence variations (PAVs) were detected in three lines. Approximately 12% and 0.5% of the total SNPs and InDels identified in three lines were located in genic regions, respectively. A total of 28 genes, harboring at least one of the large-effect SNP and/or InDel which affect the integrity of the encoded protein, were identified in the three lines. Together, 324 specific mutation genes, including 52 meiosis-related genes and 8 epigenetics-related genes were detected in neo-tetraploid rice compared to their parents. Of these 324 genes, five meiosis-related and three epigenetics-related genes displayed differential expressions during meiosis stage. Notably, 498 specific transcripts, 48 differentially expressed transposons and 245 differentially expressed ncRNAs were also detected in neo-tetraploid rice. Our results suggested that genomic structural reprogramming, DNA variations and differential expressions of some important meiosis and epigenetics related genes might be associated with high fertility in neo-tetraploid rice.