An easy to fabricate, thermally-actuated, self-regulated hydrogel valve for flow control in pneumatically driven, microfluidic systems is described. This microvalve takes advantage of the properties of the hydrogel, poly(N-isopropylacrylamide), as well as the aqueous fluid itself to realize flow control. The valve was designed for use in a diagnostic system fabricated with polycarbonate and aimed at the detection of pathogens in oral fluids at the location of the sample collection. The paper describes the construction and characterization of the hydrogel valves and their application for flow control, sample and reagent metering, sample distribution into multiple analysis paths, and the sealing of a polymerase chain reaction (PCR) reactor to suppress bubble formation. The hydrogel-based flow control is electronically addressable, does not require any moving parts, introduces minimal dead volume, is leakage and contaminant free, and is biocompatible.