Hybrid breakdown is a post-zygotic reproductive isolation that hinders genetic exchange between species or populations in both animals and plants. Two complementary recessive genes,
temperature sensitive hybrid breakdown1
(
thb1
) and
thb2
, cause hybrid breakdown in rice (
Oryza sativa
). The present study delimited the
THB1
locus to a 9.1-kb sequence, containing a single gene encoding a putative transmembrane protein with unknown functions. Haplotype analysis of
THB1
in the two core collections of 119 accessions revealed that these accessions were divided into 22 haplotypes. A test cross with
thb2
carrier showed that haplotype2 (H2) was assigned to
thb1
and was restricted to
temperate japonica
. A nonsynonymous nucleotide polymorphism (SNP) specific to H2 was identified as a causal mutation in
thb1
. A test cross with
thb1
carrier indicated that six accessions, including
temperate japonica
,
tropical japonica
, and
indica
, carried
thb2
. These results suggest that
thb1
has recently evolved in
temperate japonica
, whereas
thb2
arose in an ancient
japonica
and introgressed into the present three subgroups. Furthermore, we developed a derived cleaved amplified polymorphic sequence (dCAPS) marker to detect causal SNP in
THB1
. Our findings provide new insights into reproductive isolation and may benefit rice breeding.