Pre-harvest sprouting (PHS) is a frequently-occurring disaster that results in a serious economic loss in rice production. Breeding for PHS-resistant rice varieties has limited worldwide due to the mechanism of PHS that remains unclear. Here, nine independent mutants conferring PHS were identified by screening of an EMS (Ethylmethylsulfone) mutant library, and two of them were subjected for further study. By taking use of whole genome resequencing, SIMM method and HRM technology, two PHS mutant genes (LOC_Os03g08570 and LOC_Os07g10490) were molecularly cloned. LOC_Os03g08570 codes a phytoene desaturase (OsPDS), and LOC_Os07g10490 codes a ζ-carotene desaturase (ζ-carotene desaturase). Both were reported to be involved in the biosynthesis of carotenoid as the precursor of ABA. In summary, our data highlights the critical role of carotenoid that participates in rice dormancy. Further studies of these PHS mutants would allow revealing the mechanism of PHS and breeding for PHS-resistant rice varieties.