Genome walking (GW), a strategy for capturing of previously unsequenced DNA fragments that exist in proximity to a known sequence tag, is currently predominantly PCR-based. Recently developed PCR-based methods allow combining of sequence-specific primers with designed capturing primers capable of annealing to unknown DNA targets and offer advantages of rapidity and effectiveness of the PCR. This study presents a methodological improvement to the previously described GW technique known as Palindromic Sequence-Targeted PCR (PST-PCR). Like the PST-PCR, the new method dubbed PST-PCR v.2 relies on the targeting of capturing primers to palindromic sequences arbitrarily present in natural DNA templates. The PST-PCR v.2 consists of two rounds of the PCR. The first round uses a combination of one sequence-specific primer with one capturing (PST) primer. The second round uses a combination of a single (preferred) or two universal primers, one of which anneals to a 5’-tail attached to the sequence-specific primer and the other anneals to a different 5’-tail attached to the PST primer. The key advantage of the proposed (v.2) method is the convenience of using single universal primer with invariable sequences in GW processes involving various templates. The entire procedure takes no more than 2 - 3 hours to produce the amplified PCR fragment that contains a portion of a template flanked by the sequence-specific and capturing primers. The PST-PCR v.2 method is very suitable for simultaneous work with multiple samples for which reason the v.2 method will be applied beyond the classical task of the GW, for studies in the population genetics, in which the PST-PCR v.2 is a preferred alternative to amplified fragment length polymorphism (AFLP) or next-generation sequencing. Also, conditions for the PST-PCR v.2 are simpler to optimize as only one sequence-specific primer is used, reducing non-specific Random Amplified Polymorphic DNA (RAPD) like amplification and the formation of non-templated amplification. Importantly, like the previous version, the v.2 method is not sensitive to the template DNA’s sequence complexity or quality. This study illustrates the utility of the PST-PCR v.2 for the transposon display which a method to characterize inter- or intra-specific variability related to transposons’ integration sites. The Ac transposon sequence in the corn (Zea mays) genome was used as a sequence tag during the transposon display procedure to characterize the Ac integration sites.