l-ergothioneine levels were measured in amniotic fluid of pregnant sheep after natural mating and transfer of vitrified/thawed in-vitro produced embryos. Amniotic fluids were collected between 60 and 65 and 80–85 days of gestation and analysed by an ultra-performance liquid chromatographic (UPLC) method with fluorescence detection. l-Ergothioneine concentrations ranged between 0.23 and 9.36 μmol/L and were significantly higher in pregnancy obtained by the transfer of vitrified/thawed in-vitro produced embryos. Conversely, no significant changes in amniotic fluid l-ergothioneine concentrations were observed according to the stages of pregnancy considered in this study. These findings suggest that l-ergothioneine concentrations, are not affected as much by the gestational age, but rather by the method used to induce the pregnancy. On the whole, the measurement of l-ergothioneine in amniotic fluid could serve as a useful biomarker of oxidative stress and/or inflammatory state in pregnancy.