Two-dimensional valleytronic systems can provide information storage and processing advantages that complement or surpass those of conventional charge- and spin-based semiconductor technologies. The major challenge currently is to realize valley polarization for manipulating the valley degree of freedom. Here, we propose that valley polarization can be readily achieved in Janus single-layer MoSSe through magnetic doping, which is highly feasible in experiment. Due to inversion symmetry breaking combined with strong spin–orbit coupling (SOC), the pure single-layer MoSSe harbors an intriguing multivalleyed band structure and strong coupled spin and valley physics. After doping Cr/V, the long-sought valley polarization is successfully achieved with a remarkable energy difference of ∼0.06 eV upon switching on SOC. Furthermore, the valley polarization in Cr/V-doped single-layer MoSSe is tunable via strain engineering. Our work thus provides a promising platform for experimental studies and applications of the valleytronics.