The increasing average age emphasizes the importance of gait analysis in elderly populations. Inertial Measurement Units (IMUs) represent a suitable wearable technology for the characterization of gait by estimating spatio-temporal parameters (STPs). However, the location of inertial sensors on the human body and the associated algorithms for the estimation of gait STPs play a fundamental role and are still open challenges. Accordingly, the aim of this work was to compare three IMUs set-ups (trunk, shanks, and ankles) and correspondent algorithms to a gold standard optoelectronic system for the estimation of gait STPs in a healthy elderly population. In total, 14 healthy elderly subjects walked barefoot at three different speeds. Gait parameters were assessed for each IMUs set-up and compared to those estimated with the gold standard. A statistical analysis based on Pearson correlation, Root Mean Square Error and Bland Altman plots was conducted to evaluate the accuracy of IMUs. Even though all tested set-ups produced accurate results, the IMU on the trunk performed better in terms of correlation ( R ≥ 0.8), RMSE (0.01–0.06 s for temporal parameters, 0.03–0.04 for the limp index), and level of agreement (−0.01 s ≤ mean error ≤ 0.01 s, −0.02 s ≤ standard deviation error ≤ 0.02 s), also allowing simpler preparation of subjects and minor encumbrance during gait. From the promising results, a similar experiment might be conducted in pathological populations in the attempt to verify the accuracy of IMUs set-ups and algorithms also in non-physiological patterns.