Present work examines numerically the asymmetric behavior of hydrogen/air flame in a micro-channel subjected to a non-uniform wall temperature distribution. A high resolution (with cell size of 25 μm × 25 μm) of two-dimensional transient Navier–Stokes simulation is conducted in the low-Mach number formulation using detailed chemistry evolving 9 chemical species and 21 elementary reactions. Firstly, effects of hydrodynamic and diffusive-thermal instabilities are studied by performing the computations for different Lewis numbers. Then, the effects of preferential diffusion of heat and mass transfer on the asymmetric behavior of the hydrogen flame are analyzed for different inlet velocities and equivalence ratios. Results show that for the flames in micro-channels, interactions between thermal diffusion and molecular diffusion play major role in evolution of a symmetric flame into an asymmetric one. Furthermore, the role of Darrieus–Landau instability found to be minor. It is also found that in symmetric flames, the Lewis number decreases behind the flame front. This is related to the curvature of flame which leads to the inclination of thermal and mass fluxes. The mass diffusion vectors point toward the walls and the thermal diffusion vectors point toward the centerline. Asymmetric flame is observed when the length of flame front is about 1.1–1.15 times of the channel width.