Stress in Merino sheep can cause a reduction in quantity and quality of fine wool production. Furthermore, it has been found that environmental stress during pregnancy can negatively affect the wool follicles of the foetus. In this study we investigated the effects of early maternal shearing on lamb wool cortisol and wool phenotype traits. We conducted two experiments, the first was to determine the intra- and inter- sample variation in wool sample taken from the top- knot, and the other aim was to study the effects of shearing treatment on lamb wool cortisol and phenotype. In the first experiment, the topknot wool was collected from 10 lambs, and each sample was further divided into 4 subsamples, leading to a total of 40 subsamples. For the second experiment, we collected the topknot from the 23 lambs produced by the shearing frequency treatment ewes (once or twice shorn). The samples were then prepared and analysed using a cortisol enzyme-immunoassay in order to determine the concentration of cortisol in each of the samples. Statistical analysis for the first experiment showed that there was no significant difference between the subsamples of each wool sample (p=0.39), but there was a statistical difference between samples (p<0.001), which was to be expected. In the second experiment, there was significant difference between the lambs born to the one shearing and two shearings treatment (p=0.033), with the lambs of the twice sheared ewes having higher average wool cortisol levels (2.304 ± 0.497 ng/g; n = 22) than the ones born to once shorn ewes (1.188 ± 0.114 ng/g, n = 22). This study shows that the topknot is a reliable method of measuring cortisol in Merino lambs which could be a convenient sampling method adapted by researchers for wool sampling in lambs. Secondly, a second maternal shearing during gestation caused slightly elevated cortisol concentrations in their lambs, which warrants further investigation in a controlled study to determine if cortisol remains high during early growth and the interactions with other key endocrine, developmental and phenotypic traits.