Polymerase chain reaction (PCR) is a powerful tool in molecular biology. However, it is still a challenge to amplify large DNA fragments with complex secondary structures such as forward and/or reverse repeats and interlaced GC-rich regions. Here we describe a novel PCR protocol named nested loop PCR (NL-PCR). In this protocol, the reaction mixtures were supplemented with betaine and bovine serum albumin to enhance amplification efficiency and specificity and to overcome hindrance caused by trace amounts of contaminants. Usually, a high temperature and/or a high concentration of the additives can be applied to dissociate the secondary structure with high stability in templates for strand extension. However, Taq polymerase activity is limited to temperatures below 68 °C in the betaine-containing PCR mixtures, while a higher additive concentration may melt the newly-generated double-stranded DNA at GC-poor regions causing PCR failure. To remedy this, the NL-PCR protocol uses a temperature loop instead of the constant extension temperature used in normal PCR programs. The effectiveness of NL-PCR was confirmed by sequencing a tiling path of amplicons with an average size of 7.5 kb spanning a 120-kb contiguous sequence in a highly-repetitive region in Aegilops tauschii. Among these amplicons, one contained three forward repeats, two reverse repeats, and a region with interlaced GC-rich (> 85%) and GC-poor (< 15%) fragments. In NL-PCR, DNA fragments with different structures require different reaction conditions with specific additive concentrations and temperature loops in the PCR program. The experimental identification of optimal parameters for each fragment is labor intensive. Moreover, the design of locus-specific primers in a polyploid species is complicated by the high level of sequence identity among homoeologous genomes. To address this limitation, we designed a web-based tool for user-friendly design of locus-specific primers and determination of the appropriate PCR parameters based on target structure. This tool also contains a module to design primers for STARP (semi-thermal asymmetric reverse PCR), a recently developed genotyping method for flexible SNP and INDEL detection. NL-PCR and the web-based tool will facilitate genomic research in wheat and other species with large and complex genomes. NL-PCR and the web-based tool will be particularly useful for the targeted amplification of genes or special fragments and development of DNA markers for genomic regions hitherto untapped due to the restrictions of conventional PCR amplification.