Thermally reversible Pluronic gels have been employed as separation matrices in microfluidic devices in the analysis of biological macromolecules. The phase of these gels can be tuned between liquid and solid states using temperature to vary fluidic resistance and alter peak resolution. Although separations in thermal gels have been characterized, their effect on isotachophoresis has not. This study used fluorescein as a model analyte to evaluate isotachophoretic preconcentration as a function of thermal polymer concentration and temperature. Results demonstrated that increasing polymer concentration in microfluidic channels increased the apparent analyte concentration. A critical minimum of 10% (w/v) Pluronic was required to achieve efficient preconcentration with maximum focusing occurring in 20 and 25% polymer gels. Temperature of the thermal gel also impacted analyte focusing. Most efficient focusing was achieved at 25 degrees C with diminishing analyte accumulation at higher and lower temperatures. Under optimal conditions, isotachophoretic preconcentration increased an additional threefold simply by including thermal gels in the system. This approach can be readily implemented in other applications to increase detection sensitivity and measure low-concentration analytes within simple microfluidic devices.