Primer-BLAST is a comprehensive tool designed to facilitate the design of target-specific primers for polymerase chain reaction (PCR). The tool combines BLAST with a global alignment algorithm to ensure full primer-target alignment and detect targets with significant mismatches. It offers flexible options for adjusting specificity thresholds and primer properties, supports exon/intron placement, and excludes single nucleotide polymorphism (SNP) sites. Primer-BLAST is publicly available and can handle various input formats, including Genbank accessions, FASTA files, and pre-existing primers. It is implemented using the NCBI C++ toolkit and Primer3 C programming interface, and runs on a farm of machines at the NCBI to provide efficient service. The tool's high sensitivity and detailed alignment capabilities make it a robust solution for designing specific PCR primers, outperforming other tools like QuantPrime and PRIMEGENS in detecting potential unintended targets.Primer-BLAST is a comprehensive tool designed to facilitate the design of target-specific primers for polymerase chain reaction (PCR). The tool combines BLAST with a global alignment algorithm to ensure full primer-target alignment and detect targets with significant mismatches. It offers flexible options for adjusting specificity thresholds and primer properties, supports exon/intron placement, and excludes single nucleotide polymorphism (SNP) sites. Primer-BLAST is publicly available and can handle various input formats, including Genbank accessions, FASTA files, and pre-existing primers. It is implemented using the NCBI C++ toolkit and Primer3 C programming interface, and runs on a farm of machines at the NCBI to provide efficient service. The tool's high sensitivity and detailed alignment capabilities make it a robust solution for designing specific PCR primers, outperforming other tools like QuantPrime and PRIMEGENS in detecting potential unintended targets.