Population-based knee joint space width (JSW) assessments are promising for the prevention and early diagnosis of osteoarthritis. This study aimed to establish the statistical shape and alignment model (SSAM) of knee joints for assessing anatomic variation in knee JSW in the healthy Chinese male population. CT scans of asymptomatic knee joints of healthy male participants (n = 107) were collected for manual segmentation to create mesh samples. The as-scanned positional error was reduced by a standard processing flow of deformable mesh registration. Principal component analysis (PCA) was performed to create a tibiofemoral SSAM that was trained on all mesh samples. The anatomic variability of the JSW in the healthy Chinese male population was then assessed using the SSAM with regression analysis and 3D analysis by color-coded mapping. Almost all PCA modes had a linear influence on the anatomic variation of the medial and lateral JSW. The JSW variability within the SSAM was mainly explained by mode 1 (45.1 % of variation), demonstrating that this mode had the greatest influence on JSW variation. 3D assessment of the JSW showed that the minimum medial JSW varied from 2.76 to 3.23 mm, and its site shifted a short distance on the medial tibial plateau. The root-mean-square fitting and generalization errors of the SSAM were below 1 mm. This study will benefit the design and optimization of prosthetic devices, and may be applicable to the prevention and early diagnosis of osteoarthritis.