DIO2 was studied as a candidate gene for reproduction in sheep. Three pairs of primers were designed to detect single nucleotide polymorphisms (SNPs) of exon 2 of DIO2 in nonseasonal and seasonal reproduction breeds. The polymorphisms were detected using SeqMan II and analyzed by PCR-RFLP. The sheep DIO2 gene was cloned and its mutations were detected in five sheep breeds whose reproductive seasonality and litter sizes were different. Association analyses were performed to reveal the relationships between SNPs and reproductive seasonality as well as litter size in sheep. The results showed that a 1041-bp DNA fragment of the sheep DIO2 gene was obtained and a part of the coding sequence of 591 bp encodes 196 amino acids, having high homology with that of other mammals. Moreover, three mutations (G318T, G482C, and C885T) were detected. The test of genotype distribution in five sheep breeds showed that there were significant differences between nonseasonal and seasonal breeds for 318 and 482 loci. However, the associations between the three polymorphisms and sheep seasonal reproduction were not very distinct. Additionally, there was no significant associations between polymorphisms and litter sizes in Small Tail Han sheep. These results may provide the basic theory for further study.