Background and objectives: Statistical shape modeling (SSM) based on computerized tomography (CT) datasets has enabled reasonably accurate reconstructions of subject-specific 3D bone morphology from one or two synchronous radiographs for clinical applications. Increasing the number of radiographic images may increase the reconstruction accuracy, but errors related to the temporal and spatial asynchronization of clinical alternating bi-plane fluoroscopy may also increase. The current study aimed to develop a new approach for subject-specific 3D knee shape reconstruction from multiple asynchronous fluoroscopy images from 2, 4, and 6 X-ray detector views using a CT-based SSM model; and to determine the optimum number of planar images for best accuracy via computer simulations and in vivo experiments. Methods: A CT-based SSM model of the knee was established from 60 training models in a healthy young Chinese male population. A new two-phase optimization approach for 3D subject-specific model reconstruction from multiple asynchronous clinical fluoroscopy images using the SSM was developed, and its performance was evaluated via computer simulation and in vivo experiments using one, two and three image pairs from an alternating bi-plane fluoroscope. Results: The computer simulation showed that subject-specific 3D shape reconstruction using three image pairs had the best accuracy with RMSE of 0.52 ± 0.09 and 0.63 ± 0.085 mm for the femur and tibia, respectively. The corresponding values for the in vivo study were 0.64 ± 0.084 and 0.69 ± 0.069 mm, respectively, which was significantly better than those using one image pair (0.81 ± 0.126 and 0.83 ± 0.108 mm). No significant differences existed between using two and three image pairs. Conclusion: A new two-phase optimization approach was developed for SSM-based 3D subject-specific knee model reconstructions using more than one asynchronous fluoroscopy image pair from widely available alternating bi-plane fluoroscopy systems in clinical settings. A CT-based SSM model of the knee was also developed for a healthy young Chinese male population. The new approach was found to have high mode reconstruction accuracy, and those for both two and three image pairs were much better than for a single image pair. Thus, two image pairs may be used when considering computational costs and radiation dosage. The new approach will be useful for generating patient-specific knee models for clinical applications using multiple asynchronous images from alternating bi-plane fluoroscopy widely available in clinical settings. The current SSM model will serve as a basis for further inclusion of training models with a wider range of sizes and morphological features for broader applications.