The advent of next generation sequencing (NGS) technologies is so scale-changing that it modifies molecular diagnostics indications and induces laboratories to rethink their diagnostic strategies, until now based on the Sanger sequencing routine. Several high-throughput approaches are available from the sequencing of a gene panel, to an exome, or even a genome. In all cases, a tremendous amount of data is generated, which has to be filtered, interpreted and analyzed using powerful bioinformatics tools. In parallel, ethical considerations are raised to avoid the potential drifts of these powerful approaches. In all medical fields, and particularly in pediatrics, this new strategy offers better efficacy and faster mutation identification, allowing better support and care for patients and their families and even improving genetic counseling. In the present paper, we discuss the different NGS-based approaches and strategies as well as the issues involved in these new technologies applied to molecular diagnosis of rare diseases. Altogether, rare diseases affect more than 3 million people in France and are responsible for about one-third of childhood deaths.