The purpose of this study was to determine the effects of botulinum neurotoxin type A (BoNT-A) on vastus lateralis:​vastus medialis (VL:​VM) muscle balance, patellar tracking, and pain in patients with chronic patellofemoral (PF) pain. We recruited 13 participants (9 females, 4 males) with recalcitrant PF pain who underwent ultrasound-guided BoNT-A injections into the distal third of the VL muscle, followed by a 6-week home exercise program to strengthen their VM muscle. We imaged the participants in a C-arm computed tomography (CT) scanner before and after the intervention. We calculated VL:​VM ratios from CT images from a supine, nonweight-bearing condition. We obtained patellar tilt and bisect offset values from CT images from an upright, weight-bearing condition. We recorded functional pain scores before, immediately after, and 2–4 years after the intervention. We classified the participants into normal tracking and maltracking groups based on their patellar tilt and bisect offset values. BoNT-A with home exercise reduced VL:​VM ratio (18%;​ p < 0.001), patellar tilt (19%;​ p = 0.020), and bisect offset (5%;​ p = 0.025). Four participants classified as maltrackers before the intervention transitioned to normal tracking after the intervention. Functional pain scores improved immediately after the intervention (13%, p < 0.001) and remained improved at 2-year follow-up (12%, p = 0.011). Statement of Clinical Significance:​ This study provides new evidence in support of BoNT-A for treatment of PF pain. Classification of patients under weight-bearing conditions may identify individuals who will most benefit from a BoNT-A treatment.