Qualimap 2 is an advanced tool for quality control (QC) of high-throughput sequencing (HTS) data, offering multi-sample analysis capabilities. It allows for the simultaneous processing and comparison of multiple samples, providing a comprehensive overview of alignment data. The tool includes new modes for multi-sample analysis and RNA-seq specific bias detection. It also features a redesigned read counts QC mode and numerous user-driven improvements. Qualimap 2 is available via both graphical and command line interfaces, and its implementation is freely accessible at http://www.qualimap.org. The software is written in Java and R, and it supports various analysis modes including BAM QC, Counts QC, RNA-seq QC, and Multi-sample BAM QC. The Multi-sample BAM QC mode enables the combined QC estimation of multiple alignment files, while the RNA-seq QC mode computes metrics specific to RNA-seq data. The Counts QC mode has been completely redesigned to allow processing of multiple samples, enabling the analysis of sample grouping, outliers, and batch effects. Qualimap 2 also includes a detailed comparison of its features with other QC tools. The software has a community of users who contribute to its development and suggest new features. The tool is supported by the EU (FP7 Marie Curie Project, EIMID-IAAP, GA No. 217768 to F G.-A.). No conflicts of interest are declared.Qualimap 2 is an advanced tool for quality control (QC) of high-throughput sequencing (HTS) data, offering multi-sample analysis capabilities. It allows for the simultaneous processing and comparison of multiple samples, providing a comprehensive overview of alignment data. The tool includes new modes for multi-sample analysis and RNA-seq specific bias detection. It also features a redesigned read counts QC mode and numerous user-driven improvements. Qualimap 2 is available via both graphical and command line interfaces, and its implementation is freely accessible at http://www.qualimap.org. The software is written in Java and R, and it supports various analysis modes including BAM QC, Counts QC, RNA-seq QC, and Multi-sample BAM QC. The Multi-sample BAM QC mode enables the combined QC estimation of multiple alignment files, while the RNA-seq QC mode computes metrics specific to RNA-seq data. The Counts QC mode has been completely redesigned to allow processing of multiple samples, enabling the analysis of sample grouping, outliers, and batch effects. Qualimap 2 also includes a detailed comparison of its features with other QC tools. The software has a community of users who contribute to its development and suggest new features. The tool is supported by the EU (FP7 Marie Curie Project, EIMID-IAAP, GA No. 217768 to F G.-A.). No conflicts of interest are declared.