BackgroundNeo-tetraploid rice (NTR) is a useful new germplasm that developed from the descendants of the autotetraploid rice (ATR) hybrids. NTR showed improved fertility and yield potential, and produced high yield heterosis when crossed with indica ATR for commercial utilization. However, their classification, population structure and genomic feature remain elusive.ResultsHere, high-depth genome resequencing data of 15 NTRs and 18 ATRs, together with 38 publicly available data of diploid rice accessions, were analyzed to conduct classification, population structure and haplotype analyses. Five subpopulations were detected and NTRs were clustered into one independent group that was adjacent to japonica subspecies, which maybe the reason for high heterosis when NTRs crossed with indica ATRs. Haplotype patterns of 717 key genes that associated with yield and other agronomic traits were revealed in these NTRs. Moreover, a novel specific SNP variation was detected in the first exon of HSP101, a known heat-inducible gene, which was conserved in all NTRs but absent in ATRs, 3KRG and RiceVarMap2 databases. The novel allele was named as HSP101-1, which was confirmed to be a heat response factor by qRT-PCR, and knockout of HSP101-1 significantly decreased the thermotolerance capacity of NTR. Interestingly, HSP101-1 was also specifically expressed in the anthers of NTR at pre-meiotic and meiosis stages under optimal environment without heat stress, and its loss-of-function mutant showed significant decrease in fertility of NTR.ConclusionThe construction of first genomic variation repository and the revelation of population structure provide invaluable information for optimizing the designs of tetraploid rice breeding. The detection of specific genomic variations offered useful genomic markers and new directions to resolve high fertility mechanism of NTR.