Introduction: Anterior Cruciate Ligament Reconstruction (ACLR) is crucial for knee stability in ACL-injured individuals and for resuming pre-injury physical activities. Despite advancements, achieving symmetrical movement patterns during rehabilitation, particularly in stair negotiation, poses challenges. This study examines lower limb kinematics during stair negotiation at various rehabilitation stages post-ACLR, employing inertial measurement units (IMUs) and Statistical Parametric Mapping (SPM) for in-depth analysis outside the laboratory. Methods: This cohort study longitudinally assessed stair ambulation kinematics in patients aged 18-40, three (n=26) and five months (n=18) post-ACLR, using IMUs to track sagittal plane movement during stair ascent and descent. The participants ambulated on a flight of 20 stairs outside the laboratory. Results: At three months post-ACLR, the injured knee was less flexed compared to the contralateral knee during stair ascent (mean difference = -11.3, CI [-14.4, -8.1], p<.001) and descent (mean difference = -6.3, CI [-10.2, -2.4], p=.002). SPM analysis identified clusters where the injured knee showed decreased flexion at 0-35% and 87-99% of the stair ascent cycle (p<.005). By five months, flexion differences during ascent improved (mean difference = -4.7, CI [-8.1, -1.4], p=.008), but significant asymmetry persisted, with decreased flexion at 10-32% of the cycle during ascent and 20-29% during descent (p<.017). Improvements between three and five months were observed in knee flexion during ascent (mean increase = 6.1, p<.001) and descent (mean increase = 9.3, p=.004). Ankle and hip joint movements also exhibited persistent asymmetries, with minimal improvements over time. Conclusions: Persistent lower limb kinematic asymmetries remain five months post-ACLR during stair ascent and descent.