Human reference genome, the most important outcome of the Human Genome Project, has led to revolutionary developments in many fields of human genetics including genetic diagnostics. However, human genome project was lacked in displaying inter-individual genomic variability. This vacuum was filled by widespread usage of second generation sequencing, also known as next-generation sequencing. Today, the vast information about human genome diversity obtained by such sequencing methods are accessible to clinicians and researchers through several databases. Second generation sequencing is a method involving the simultaneous sequencing of small DNA fragments in parallel, together with combination and analysis of the sequence information through bioinformatic tools. Currently, this method allows for sequencing of a whole human genome (WGS) in one single run. However, whole exome sequencing (WES) focusing on only protein/RNA coding exonic regions or other limited sequencing methods are in more common use. In this chapter, the principles making second generation sequencing technologies available, analysis of second generation sequencing, constraints and superiority of the technology will be explained. Additionally, the impact of second generation sequencing with a special focus on the field of medicine will be mentioned.