The investigation of the in-plane shear behavior of prepreg is crucial for understanding the generation of wrinkles of preforms in advanced composite manufacturing processes, such as automated fiber placement and thermoforming. Despite this significance, there is currently no standardized test method for characterizing uncured unidirectional (UD) prepreg. This paper introduces a ±45° off-axis tensile test designed to assess the in-plane shear behavior of UD carbon fiber-reinforced epoxy prepreg (CF/epoxy). Digital image correlation (DIC) was employed to quantitatively track the strains in three dimensions and the shear angle evolution during the stretching process. The influences of the temperature and stretching rate on the in-plane shear behavior of the prepreg were further investigated. The results reveal that four shear characteristic zones and wrinkling behaviors are clearly distinguished. The actual in-plane shear angle is significantly lower than the theoretical value due to fiber constraints from both the in-plane and out-of-plane aspects. When the off-axis tensile displacement (d) is less than 15.6 mm, the ±45° specimens primarily exhibit macroscale in-plane shear behavior, induced by interlaminar interface shear between the +45° ply and −45° ply at the mesoscale. The shear angle increases linearly with the d. However, when d > 15.6 mm, fiber squeezing and wrinkling begin to occur. When d > 29 mm, the in-plane shear disappears in the completely sheared zone (A). The reduction in the resin viscosity of the CF/epoxy prepreg caused by increased temperature is identified as the primary factor in lowering the in-plane shear force resistance, followed by the effect of the increasing resin curing degree. Higher shear rates can lead to a substantial increase in shear forces, eventually causing cracking failure in the prepreg. The findings demonstrate the feasibility of the test method for predicting and extracting uncured prepreg in-plane shear behaviors and the strain-rate and temperature dependency of the material response.