The integration of next-generation sequencing (NGS) has elevated organoid research significantly and was instrumental in making patient-derived organoids (PDOs) a promising tool for biomarker discovery, drug response analysis in vivo, and personalized treatment strategies. On one hand, high-throughput capabilities have made NGS invaluable in comparing organoids with parental tumors they were derived from, assess genetic stability over multiple organoid passages, and characterize rare subpopulations in organoids. On the other, organoids have the capability to increase sequencing depth and sensitivity compared to necrosis-prone tumor biopsies. This review highlights the applications, advantages, and limitations of NGS methods in genomics, transcriptomics, and epigenomics when applied to organoid-based cancer studies.