We developed a modular software package SNPbox that automates and standardizes the generation of PCR primers and is used in the strategy for constructing single nucleotide polymorphisms (SNPs) maps. In this strategy, the focus of primer design can be either on the validation of annotated public SNPs or on the SNP discovery in exon regions or extended genomic regions, both by resequencing. SNPbox relies on Primer3 for the primer design and combines this program with other publicly available software tools such as BLAST, Spidey and RepeatMasker, and newly developed algorithms. Primer conditions were chosen such that PCR amplifications are uniform for each PCR amplicon facilitating the use of high-throughput genetic platforms. SNPbox can also be used for the design of primer sets for mutation analysis, STR marker genotyping and microarray oligo design. Of the 2500 primer sets designed by SNPbox, 95% successfully amplified genomic DNA under uniform PCR conditions.