AbstractConventional diffusion‐weighted imaging (DWI) sequences employing a spin echo or stimulated echo sensitize diffusion with a specific b‐value at a fixed diffusion direction and diffusion time (Δ). To compute apparent diffusion coefficient (ADC) and other diffusion parameters, the sequence needs to be repeated multiple times by varying the b‐value and/or gradient direction. In this study, we developed a single‐shot multi‐b‐value (SSMb) diffusion MRI technique, which combines a spin echo and a train of stimulated echoes produced with variable flip angles. The method involves a pair of 90° radio frequency (RF) pulses that straddle a diffusion gradient lobe (GD), to rephase the magnetization in the transverse plane, producing a diffusion‐weighted spin echo acquired by the first echo‐planar imaging (EPI) readout train. The magnetization stored along the longitudinal axis is successively re‐excited by a series of n variable‐flip‐angle pulses, each followed by a diffusion gradient lobe GD and a subsequent EPI readout train to sample n stimulated‐echo signals. As such, (n + 1) diffusion‐weighted images, each with a distinct b‐value, are acquired in a single shot. The SSMb sequence was demonstrated on a diffusion phantom and healthy human brain to produce diffusion‐weighted images, which were quantitative analyzed using a mono‐exponential model. In the phantom experiment, SSMb provided similar ADC values to those from a commercial spin‐echo EPI (SE‐EPI) sequence (r = 0.999). In the human brain experiment, SSMb enabled a fourfold scan time reduction and yielded slightly lower ADC values (0.83 ± 0.26 μm2/ms) than SE‐EPI (0.88 ± 0.29 μm2/ms) in all voxels excluding cerebrospinal fluid, likely due to the influence of varying diffusion times. The feasibility of using SSMb to acquire multiple images in a single shot for intravoxel incoherent motion (IVIM) analysis was also demonstrated. In conclusion, despite a relatively low signal‐to‐noise ratio, the proposed SSMb technique can substantially increase the data acquisition efficiency in DWI studies.