Many sequence-alignment tools were developed to discover single nucleotide polymorphisms (SNPs) derived from resequencing in genomic regions. Whether an identified SNP is indeed a novel SNP or is already contained in dbSNP is often difficult to answer. Here, we describe a freely available software, Seq-SNPing, which is a Java-based software for SNP discovery, and ID identification and editing and visualizating of sequence alignments. It is easy to use, fast, and provides an accurate method for searching and organizing SNP IDs from multiple sequence inputs, thereby greatly facilitating genetic studies. Seq-SNPing provides SNP identification by selecting any range of unaligned or aligned sequences in sequences that are similar. SNP IDs in the National Center for Biotechnology Information (NCBI) or user-defined SNPs within a selected sequence can be identified by Seq-SNPing. Information needed for SNP-RFLP (restriction fragment length polymorphism) genotyping is provided, such as SNP-REs (restriction enzymes), the sequence trimmer, sequence finder, BLAST (Basic Local Alignment Search Tool), SNP-BLAST, UCSC BLAT (BLAST-like alignment tool), RE mining, antisequencer (Anti-seq), and T(m) (melting temperature)/GC% of selected sequence. The thresholds for SNP calling are adjustable by selecting the height of the peak for each nucleotide representative curve in the chromatogram. Therefore, Seq-SNPing can discover SNPs and identify SNP IDs in both sequence text and chromatogram files in a fast and reliable way. The software is fully compatible with Microsoft Windows. The program and user manual are available at http://bio.kuas.edu.tw/Seq-SNPing for download.