At advanced knee osteoarthritis (OA) stages subchondral trabecular bone (STB) is altered. Lower limb alignment plays a role in OA progression and modify the macroscopic loading of the medial and lateral condyles of the tibial plateau. How the properties of the STB relate to alignment and OA stage is not well defined. OA stage (KL scores 2-4) and alignment (HKA from 17° Varus to 8° Valgus) of 30 patients were measured and their tibial plateau were collected after total knee arthroplasty. STB tissue elastic modulus, bone volume fraction (BV/TV) and trabecula thickness (Tb.Th) were evaluated with nanoindentation and µCT scans (8.1 µm voxel-size) of medial and lateral samples of each plateau. HKA and KL scores were statistically significantly associated with STB elastic modulus, BV/TV and Tb.Th. Medial to lateral BV/TV ratio correlated with HKA angle (R = −0.53, p = 0.016), revealing a higher ratio for varus than valgus subjects. STB properties showed lower values for KL stage 4 patients. Tissue elastic modulus ratios and BV.TV ratios were strongly correlated (R = 0.81, p < 0.001). Results showed that both micromechanical and microarchitectural properties of STB are affected by macroscopic loading at late stage knee OA. For the first time, a strong association between tissue stiffness and quantity of OA STB was demonstrated.