Hormonal assessment tools are important for determining the reproductive success of pro- duction animals. This study used non-invasive wool assessment to quantify changes in pro- gesterone and cortisol levels in reproducing female merino sheep. Wool samples were collected from a group of n = 46 maiden merino ewes (22–25 months old), naturally joined under natural light conditions in southern New South Wales (NSW), Australia. Three shear- ing opportunities were conducted as part of standard on-farm management practices. The wool samples were collected at three different dates during 2017, January (prior to rams being put out with the mob and to provide a baseline level since previous shearing in May 2016), September (during very late stages of gestation–approximately 2 weeks prior to par- turition) and December (ewes had given birth and ~2-month-old lambs were at foot). Analy- sis of cortisol and progesterone was conducted concurrently from the same sample of wool. The hormones in wool samples quantified using commercially available cortisol and proges- terone enzyme-immunoassay kits. Wool cortisol concentrations increased significantly (p = 3.04E-14) from pre-joining in January (1.33±0.12 ng/g) to late gestation in September (3.59 ±0.12 ng/g). Concentration of wool cortisol post-lambing in December (3.27±0.14 ng/g) did not decline significantly (p = 0.124) after gestation however remained significantly higher (p = 3.82E-10) than pre-joining levels. Wool progesterone (PG) concentrations increased sig- nificantly (p = 1.83E-33) from pre-joining (0.04±0.005 ng/g) in January to late gestation in September (5.53±0.13 ng/g) with a significant (p = 5.44E-59) decline observed in December (0.05±0.003 ng/g) to post- pregnancy concentrations. No significant difference was shown between pre-joining and post lambing PG concentrations (p = 0.057). Our results showed that non-invasive assessment of hormones in Merino sheep wool reflected significant increase in both cortisol and progesterone guided by pregnancy.