DIO3 and DIO3OS are two imprinted genes identified in mouse and humans. The DIO3 gene, which encodes for the type 3 deiodinase, is preferentially expressed from the paternal allele, while the DIO3OS transcript is transcribed in opposite orientation to DIO3, multiple noncoding and alternatively splicing isoforms from maternal allele. In this study, the five splice variants of DIO3OS were identified in Holstein cattle and had complex, tissue-specific expression patterns observed in eight tissues, including heart, liver, spleen, lung, kidney, muscle, fat and brain. In the G+C rich region, upstream from the cattle DIO3 gene, there were three small conserved regions and some promoter elements similar to those observed in mouse and humans. An allele-specific expression analysis-based SNP method revealed that DIO3 and DIO3OS genes exhibited monoallelic expression in the eight tissues, indicating that DIO3 and DIO3OS are imprinted in cattle.