Abstract We investigate the effect of structural and substitutional defects in Janus MoSSe and the Janus alloys MoS 2(1− x ) Se 2 x by a comprehensive analysis. Distinct Raman signatures are associated with various defect types and densities, mirroring the evolution from MoSe 2 to Janus alloys to ideal Janus MoSSe. By the corresponding stoichiometrical and structural changes, the band gap can be tuned from 1.50 eV up to 1.68 eV at room temperature. Electrical characterization in a field effect device uncovers the impact of defects on conductivity, mobility (up to 2.42 × 10 −3 cm 2 V −1 s −1 ), and threshold voltages. A decrease of n-type doping of 5.3 × 10 11 cm −2 in Janus MoSSe compared to the Janus alloy points towards an increased work function and a reduction of defects. Our findings deepen the understanding of defect physics in 2D Janus materials and pave the way for tailored defect engineering strategies for advanced (opto-)electronic applications.