Polymerase chain reaction is the most common laboratory method used in diagnosis of infectious diseases. It still requires cold-chain transportation and storage of reaction components, adequate instrumentation, as well as a skilled specialist during the preparation of reaction mixes. This study was focused on the evaluation of pre-prepared freeze-dried PCR mixes for virus detection in ready-to-use form. PCR mixes with new commercial glycerol-free DNA polymerase, cryoprotectants as trehalose, dextran or mannitol and other PCR components were lyophilized and stored at room temperature for several months. Our study revealed that 10% trehalose, 5% dextran and their combination stabilized PCR mixes for the detection of porcine circovirus 2 (PCV2) and hepatitis E virus (HEV) for 3 months. The freeze-dried PCR mixes detected those viruses in concentrated and 10 to 100-fold diluted clinical samples. The lyophilized PCR mixes containing commercially available glycerol-free DNA polymerase stabilized with sugar cryoprotectants were suitable for the preparation of diagnostic PCR kits.