Next generation sequencing is a vernacular method of high parallel sequencing that produces genomic data to sample genetic diversity within and between germplasm pools. The new generation technique has emerges considerably with many advantages over indigenous sequencing techniques, i.e., higher throughput, faster turnaround time, longer read lengths, higher consensus accuracy to enable rare variant detection, small amounts of sample required (in third generation sequencing single molecule needed) for sequencing at low cost. The chief NGS method include second and third generation sequencing, among which 454 (Rocha), Illumina and Ion torrent is based on principle of sequencing by synthesis, while solid sequencing rely on sequencing by ligation. The third generation sequencing is superior, as no clonal amplification leads to larger genome sequence in less time. NGS have substantial implications in crop genetics and breeding, facilitating trait mapping and making marker-assisted breeding more feasible. In animals, genomic, transcriptomic knowledge obtain through NGS help in field of biomedical and life sciences such as treatment of human diseases, development of alternative bio fuels, enhancement of crop yield, ensuring food safety, forensics, etc. This chapter highlights the principle, sequencing chemistry along with brief comparison of these high throughput sequencing technologies and recent advancing platform available for these sequencing