The article discusses the latest improvements and enhancements to Primer3, a widely used software for primer design in PCR applications. The authors highlight several key advancements, including more accurate thermodynamic models for predicting melting temperatures and reducing the formation of hairpins or dimers. They also introduce new features for precise control over primer placement, leveraging whole-genome sequences to improve specificity. Additionally, Primer3 now supports saving and reusing parameter settings, enhancing usability and integration with other software. The core code has been modularized, and a C/C++ programming library, libprimer3, has been developed to facilitate direct integration with third-party programs. These improvements position Primer3 as a robust tool for genome-scale data analysis in the future.The article discusses the latest improvements and enhancements to Primer3, a widely used software for primer design in PCR applications. The authors highlight several key advancements, including more accurate thermodynamic models for predicting melting temperatures and reducing the formation of hairpins or dimers. They also introduce new features for precise control over primer placement, leveraging whole-genome sequences to improve specificity. Additionally, Primer3 now supports saving and reusing parameter settings, enhancing usability and integration with other software. The core code has been modularized, and a C/C++ programming library, libprimer3, has been developed to facilitate direct integration with third-party programs. These improvements position Primer3 as a robust tool for genome-scale data analysis in the future.