Retinitis Pigmentosa (RP) is a major cause of heritable human blindness with a high genetic heterogeneity. It is characterized by the initial degeneration of rod photoreceptors followed by cone photoreceptors. RP is also a prominent reason of visual impairment, by a global prevalence of 1:4000. RP is usually specified with nyctalopia in puberty, followed by concentric visual ﬁeld loss, that reﬂects the main impairment of rod photoreceptors; later in the life, as disease progresses, because of cone dysfunction, central vision loss also occurs. A precise molecular diagnosis is crucial for disease characterization and clinical prognosis. DNA sequencing is a powerful tool for deciphering various causes of different human diseases. The arrival of next-generation sequencing (NGS) technologies has diminished sequencing cost and considerably augmented the throughput, making whole-genome sequencing (WGS) a conceivable way for obtaining comprehensive genomic data and a more precise clinical decision. Nevertheless, the advantages gained from NGS technologies are among a number of challenges that must be sufficiently addressed before this technique can be altered from an investigation tools to a helpful method in routine clinical practices. This article aims to provide an overview about NGS technology and its related platforms. The challenges in the analysis and choosing an appropriate NGS method likewise their potential applications in clinical diagnosis are also discussed. The merit of such technique has been reflected in some recent studies where it is shown that using NGS and molecular information could help with clinical diagnosis, providing potential treatment options or changes, up-to-date family counselling and management.