Joule heating is a significant problem for microfluidic chips with electrokinetically driven flows. In this paper, we will present the modeling results of the Joule heating of a Polymethylmethacrylate (PMMA) polymer separation chip using both experimental and computational methods. The temperature distributions on the surface of the chip were measured by an advanced thermograph system. The numerical study was carried out using the multiphysics computational fluid dynamics (CFD) package CFD-Ace+. Different solutions and operating conditions were studied. Both the measurements and CFD data revealed that the heat generation was approximately uniform and the subsequent temperature increase was also uniform along the channel except for regions near the liquid ports. The highest temperature increase was observed along the centerline of the channel and the temperature reduced significantly away from the channel. At an electrical field of 45kV/m, the Joule heating effect was negligible for the solution used, even though at such a high electric field significant heating effect has been observed for micro capillary flows in literature. At a higher electrical field (68-120kV/m), the Joule heating could cause an increase of temperature of up to 40°C.