OsWRKY70 is a key transcription factor in rice response to low-temperature stress. However, the genetic mechanism by which its natural variation regulates cold tolerance in rice remains unclear. This study analyzed the haplotype composition, population differentiation, and correlation with cold tolerance of the OsWRKY70 coding region based on 3K rice genomes project. The results showed that 26 SNPs exist in the OsWRKY70 coding region, constituting 9 haplotypes, among which Hap1 (56.54%) and Hap2 (42.24%) were the dominant haplotypes. Population genetic analysis showed significant differentiation of this gene between indica and japonica rice subspecies (Fst = 0.598). Hap1 was identified as the characteristic haplotype of indica rice (accounting for 83.10% of indica rice), while Hap2 was the characteristic haplotype of japonica rice (accounting for 98.71% of japonica rice). They contributed 99.98% of the population differentiation index, indicating that this locus is an important site for differentiation between indica and japonica rice. Geographical distribution analysis revealed that Hap1 is mainly distributed in tropical and southern subtropical regions, while Hap2 is more common in high-latitude, high-altitude areas and regions with frequent low temperatures. Cold tolerance phenotypic identification showed that the average survival rate of Hap2 (51.86%) was significantly higher than that of Hap1 (4.73%), indicating that Hap2 is a dominant cold tolerance haplotype, and this cold tolerance effect is more significant in the genetic context of japonica rice. This study clarified the association between natural variation in OsWRKY70 and differentiation between indica and japonica rice varieties, as well as cold tolerance. Five non-synonymous SNPs found between Hap2 and Hap1 provide a theoretical basis for genetic improvement of cold tolerance and the development of related molecular markers.