Qualimap 2 is an advanced bioinformatics tool for quality control (QC) of high-throughput sequencing (HTS) data, designed to address the limitations of existing QC tools that often only handle single samples. This version introduces new features for multi-sample analysis, allowing simultaneous processing and comparison of multiple samples. The tool includes four analysis modes: *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, facilitating the examination of consistency and detection of outliers among samples. The *RNA-seq QC* mode provides specific metrics for RNA-seq data, such as per-transcript coverage and junction sequence distribution. The *Counts QC* mode has been redesigned to handle multiple samples, allowing the analysis of read counts from multiple biological replicates. Qualimap 2 is available for free at http://www.qualimap.org and is maintained by a community of users who contribute to its development.Qualimap 2 is an advanced bioinformatics tool for quality control (QC) of high-throughput sequencing (HTS) data, designed to address the limitations of existing QC tools that often only handle single samples. This version introduces new features for multi-sample analysis, allowing simultaneous processing and comparison of multiple samples. The tool includes four analysis modes: *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, facilitating the examination of consistency and detection of outliers among samples. The *RNA-seq QC* mode provides specific metrics for RNA-seq data, such as per-transcript coverage and junction sequence distribution. The *Counts QC* mode has been redesigned to handle multiple samples, allowing the analysis of read counts from multiple biological replicates. Qualimap 2 is available for free at http://www.qualimap.org and is maintained by a community of users who contribute to its development.