PURPOSE: This study aimed to identify the discriminating kinematic gait characteristics between individuals with acute and chronic patellofemoral pain (PFP) and healthy controls. METHODS: Ninety-eight runners with PFP (39 male, 59 female) and 98 healthy control runners (38 male, 60 female) ran on a treadmill at a self-selected speed while three-dimensional lower limb kinematic data were collected. Runners with PFP were split into acute (n = 25) and chronic (n = 73) subgroups on the basis of whether they had been experiencing pain for less or greater than 3 months, respectively. Principal component analysis and linear discriminant analysis were used to determine the combination of kinematic gait characteristics that optimally separated individuals with acute PFP and chronic PFP and healthy controls. RESULTS: Compared with controls, both the acute and chronic PFP subgroups exhibited greater knee flexion across stance and greater ankle dorsiflexion during early stance. The acute PFP subgroup demonstrated greater transverse plane hip motion across stance compared with healthy controls. In contrast, the chronic PFP subgroup demonstrated greater frontal plane hip motion, greater knee abduction, and reduced ankle eversion/greater ankle inversion across stance when compared with healthy controls. CONCLUSIONS: This study identified characteristics that discriminated between individuals with acute and chronic PFP when compared with healthy controls. Certain discriminating characteristics were shared between both the acute and chronic subgroups when compared with healthy controls, whereas others were specific to the duration of PFP.