Purpose The longitudinal changes in alignment and structure, including the joint line and cortical bone thickness (CBT) of the femur and tibia, and knee phenotype in patients with knee osteoarthritis (OA) remain unknown. The aim of this retrospective study was to clarify the longitudinal changes in matched healthy subjects. Methods The follow-up Matsudai Knee Osteoarthritis Survey was administered between 23 and 28 years. This study included 285 healthy knees from 235 females with an average age of 53 ± 6 years at baseline. The non-OA individuals, with an average age of 79 ± 4 years, were divided into three groups at baseline according to their follow-up radiographic results [the non-OA (n = 52), early OA (n = 131), and advanced OA groups (n = 102)]. Changes in alignment, joint line, CBT, and knee phenotype were assessed at baseline and at follow-up using standing anteroposterior radiographs. Results This study showed signiicant varus changes in the alignment (p < 0.001) and tibial and femoral joint line parameters (p < 0.05) in the OA group. Decreased CBT and increased mediolateral CBT ratios were observed in all groups (p < 0.001). The knee phenotypes in the OA groups were changed to varus angles, especially in the alignment and tibial joint line. Conclusions The longitudinal changes of knee phenotypes in alignment and structure (CBT and joint line) from baseline to follow-up were shown in the OA groups. In addition, alignment and tibial structural factors at baseline are useful in predicting the incidence of knee OA in daily practice. Levels of evidence III.