The imprinted isoform of the Mestgene in mice is involved in key mammalian traits such as placental and fetal growth, maternal care and mammary gland maturation. MEST has a distinct promoter differentially methylated region (DMR) in eutherian mammals but in marsupials, while MEST was thought to be imprinted, it had no DMR. In this study, we examined similarities and differences in the MEST gene locus across mammals using a marsupial, the tammar wallaby, a monotreme, the platypus, and a eutherian, the mouse, to investigate how MEST imprinting evolved in mammals. By con rming the presence of the short isoform in all mammalian groups (which is imprinted in eutherians), this study suggests that an alternative promoter for the short isoform has evolved at the MESTgene locus in the common ancestor of mammals. In the tammar, the short isoform of MEST shared the putative promoter CpG island with an antisense lncRNA previously identi ed in humans and an isoform of a neighbouring gene CEP41. The antisense lncRNA was expressed in tammar sperm, as seen in humans. This suggested that the conserved lncRNA might play an important part in the establishment of MEST imprinting in therian mammals, but it was not imprinted in the tammar. In contrast to previous studies, this study showed that MEST is non-imprinted and mono-allelically expressed in marsupials. This suggests that selection of MEST imprinting in eutherians must have occurred after the marsupial-eutherian split with the acquisition of a key epigenetic imprinting control region, the DMR.