The Genome Analysis Toolkit (GATK) is a widely used software suite for identifying and annotating genomic variants from high-throughput sequencing data. Originally developed by the Broad Institute, GATK has become a cornerstone in genomic research thanks to its modular design, reproducible workflows, and integration with cloud platforms. Key tools such as HaplotypeCaller, Mutect2, and Funcotator enable researchers to detect germline and somatic variants, assess functional impact, and process large datasets with efficiency and accuracy. Its workflows support best practices for variant calling—both in human and non-model organisms—which makes it valuable across a broad range of disciplines ranging from clinical genomics to evolutionary biology. With the increasing scale and accessibility of sequencing technologies, tools like GATK play a critical role in transforming raw data into meaningful biological insights. This review outlines the toolkit’s major components, common use cases, and practical considerations, offering a foundation for researchers looking to implement or better understand GATK in their own analyses.