The γ-ray emission originating from in-flight annihilation (IA) of positrons is a powerful observable for constraining high-energy positron production from exotic sources. By comparing diffuse γ-ray observations of INTEGRAL, COMPTEL, and EGRET to theoretical predictions, we set the most stringent constraints on electrophilic feebly interacting particles, thereby proving IA as a valuable probe of new physics. In particular, we extensively discuss the case of MeV-scale sterile neutrinos, where IA sets the most stringent constraints, excluding |Uμ4|2≳10-13 and |Uτ4|2≳2×10-13 for sterile neutrinos mixed with μ and τ neutrinos, respectively. These constraints improve existing limits by more than an order of magnitude. We briefly discuss the application of these results to a host of exotic positron sources such as dark photons, axionlike particles, primordial black holes, and sub-GeV dark matter.