In this paper, we present a novel PCR method, termed SiteFinding-PCR, for gene or chromosome walking. The PCR was primed by a SiteFinder at a low temperature, and then the target molecules were amplified exponentially with gene-specific and SiteFinder primers, and screened out by another gene-specific primerand a vector primer.However, non-target molecules could not be amplified exponentially owing to the suppression effect of stem-loop structure and could notbescreenedout. This simplemethodproved to be efficient, reliable, inexpensive and time-saving, andmay be suitable for themolecules for which genespecific primers are available. More importantly, large DNA fragments can be obtained easily using this method. To demonstrate the feasibility and efficiency of SiteFinding-PCR, we employed this method to do chromosomewalkingandobtained 16positive results from 17 samples.