Loss of function of duplicated genes plays an important role in the evolution of postzygotic reproductive isolation. The widespread occurrence of gene duplication followed by rapid loss of function of some of the duplicate gene copies suggests the independent evolution of loss-of-function alleles of duplicate genes in divergent lineages of speciation. Here, we found a novel loss-of-function allele of S27 in the Asian annual wild species Oryza nivara, designated S27-niv (s), that leads to F(1) pollen sterility in a cross between O. sativa and O. nivara. Genetic linkage analysis and complementation analysis demonstrated that S27-niv (s) lies at the same locus as the previously identified S27 locus and S27-niv (s) is a loss-of-function allele of S27. S27-niv (s) is composed of two tandem mitochondrial ribosomal protein L27 genes (mtRPL27a and mtRPL27b), both of which are inactive. The coding and promoter regions of S27-niv (s) showed a number of nucleotide differences from the functional S27-T65 (+) allele. The structure of S27-niv (s) is different from that of a previously identified null S27 allele, S27-glum (s), in the South American wild rice species O. glumaepatula, in which mtRPL27a and mtRPL27b are absent. These results show that the mechanisms for loss-of-function of S27-niv (s) and S27-glum (s) are different. Our results provide experimental evidence that different types of loss-of-function alleles are distributed in geographically and phylogenetically isolated species and represent a potential mechanism for postzygotic isolation in divergent species.