DNA sequencing is one of the great valuable techniques in molecular biology, which can be used to detect the sequence of nucleotides in a DNA fragment. The high-throughput sequencing known as Next generation sequencing (NGS) revolutionized genomic research and molecular biology; therefore, the whole human genome can be sequenced with a low cost in several days. NGS technology is similar to the traditional method, Sanger, which detects small DNA fragments by emitted signals at the time of synthesis of each fragment (from the DNA template), but the difference is that NGS can determine the massive simultaneous sequencing in a few days with high accuracy and the results are directly detected without the need for electrophoresis. In fact, NGS technology combines a variety of steps such as sample preparation, fragmentation of the sample of the studied genome, attachment of adapter to the ends of the fragments, imaging, and data analyses. In recent years, NGS technology continuously expanded the range of applications in different fields by reducing costs, increasing rates, and improving the quality of the data. The current review provided the potential applications of the NGS technology by emphasizing the diagnosis of the genetic diseases, identification of several types of cancers, prenatal screening, epigenetic modifications, personalized medicine, and identification of pathogens.