Objectives: The purpose of this study was to quantify the tissue level mechanical properties of cortical bone of skeletally immature (~five-month-old) Beagle dogs and compare them to data from mature dogs measured in a previous study. Methods: Eight femoral cross sectional specimens (two bone sections / dog) were obtained from four skeletally immature dogs. A pair of calcein bone labels were administered intravenously to the dogs to mark sites of active mineralization prior to euthanasia. Prepared bone specimens were placed in a nano- indenter specimen holder and the previously identified calcein labelled osteons were located. Labelled (n = 128) and neighbouring unlabelled (n = 127) osteons in skeletally immature femurs were examined by instrumented indentation testing. Indents were made to a depth of 500 nm at a loading rate of 10 nm/s. Indentation modulus (IM) and hardness (H) were obtained. Results: The overall IM of the cortical bone in the skeletally mature groups was significantly greater than in the immature group (p = 0.0011), however overall H was not significantly different. The differences between the groups in IM were significant for the unlabelled osteons (p = 0.001), but not for the labelled osteons (p = 0.56). Conclusion : There are differences in the IM of unlabelled osteons in skeletally immature and mature groups of Beagle dogs. In contrast to whole bone mechanical tests, where there are obvious differences between growing and mature bones, there are only small differences in the micro-mechanical properties.