ABSTRACT The advent of massive parallel sequencing is rapidly changing the strategies employed for the genetic diagnosis and research of rare diseases that involve a large number of genes. So far it is not clear whether these approaches perform significantly better than conventional single gene testing as requested by clinicians. The current yield of this traditional diagnostic approach depends on a complex of factors that include gene‐specific phenotype traits, and the relative frequency of the involvement of specific genes. To gauge the impact of the paradigm shift that is occurring in molecular diagnostics, we assessed traditional Sanger‐based sequencing (in 2011) and exome sequencing followed by targeted bioinformatics analysis (in 2012) for five different conditions that are highly heterogeneous, and for which our center provides molecular diagnosis. We find that exome sequencing has a much higher diagnostic yield than Sanger sequencing for deafness, blindness, mitochondrial disease, and movement disorders. For microsatellite‐stable colorectal cancer, this was low under both strategies. Even if all genes that could have been ordered by physicians had been tested, the larger number of genes captured by the exome would still have led to a clearly superior diagnostic yield at a fraction of the cost. In order to determine the impact of exome sequencing in molecular diagnostics, the diagnostic yields for traditional Sanger‐based sequencing and exome sequencing followed by targeted bioinformatics analysis were compared for five genetically heterogeneous disorders. We find that exome sequencing has a much higher diagnostic rate than Sanger sequencing for four of these, being deafness, blindness, mitochondrial disease, and movement disorders. We conclude that exome sequencing is applicable to a broad range of heterogeneous genetic diseases, being an overall effective choice, with respect to both diagnostic yield as well as costs.