The specific complementary association due to hydrogen bonding of single stranded nucleic acids is referred to as annealing. Successful primer annealing is of critical importance in a polymerase chain reaction (PCR). In this study, the effect of gradually increased and decreased annealing temperatures on PCR reactions were investigated. In the annealing tests, the isolates of Plum bark necrosis stem pitting associated virus (PBNSPaV), Apricot latent virus (ApLV), Apple scar skin viroid (ASSVd) and Potato virus Y (PVY) were used as reference isolates. The effects of annealing temperature alterations were tested by nested reverse transcriptase polymerase chain reaction (nested-RT-PCR) for PBNSPaV and RT-PCR for ApLV, ASSVd and PVY. The PCR products were analyzed and evaluated by polyacrylamide gel electrophoresis (PAGE). Gradient temperatures (2 to 4 degrees C) below and above annealing temperature resulted in increased primer annealing by both methods. Higher gradients (8-12 degrees C) never resulted in appreciable yields of a unique PCR product, as the likelihood of primer annealing was reduced. Both high and low gradients of annealing temperature gave the yield in PCR reactions, confirming flexible nature of annealing temperature.