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Abstract Instability occurs in the electrokinetic flow of fluids with conductivity and/or permittivity gradients if the applied electric field is beyond a critical value. Understanding such an electrokinetic instability is significant for both improved transport (via the suppressed instability) and enhanced mixing (via the promoted instability) of liquid samples in microfluidic applications. This work presents the first study of Joule heating effects on electrokinetic microchannel flows with conductivity gradients using a combined experimental and numerical method. The experimentally observed flow patterns and measured critical electric fields under Joule heating effects to different extents are reasonably predicted by a depth‐averaged numerical model. It is found that Joule heating increases the critical electric field for the onset of electrokinetic instability because the induced fluid temperature rise and in turn the fluid property change (primarily the decreased permittivity) lead to a smaller electric Rayleigh number.