Congenital myasthenic syndromes (CMSs) are clinically and genetically heterogeneous disorders characterized by a neuromuscular transmission defect. Even though CMSs are genetic disorders, they are highly treatable, and the appropriate drug treatment depends on the underlying genetic defect. This highlights the importance of genetic testing in CMS. In recent years, the molecular basis of CMS has constantly broadened and disease-associated mutations have been identified in 14 genes encoding proteins of the neuromuscular junction. In the dawn of novel sequencing strategies, we report on our 14-year experience in traditional Sanger-based mutation screening of a large cohort of 680 independent patients with suspected CMS. In total, we identified disease-causing mutations in 299 patients (44%) of patients in various known CMS genes, confirming the high degree of genetic heterogeneity associated with the disease. Apart from four known founder mutations, and a few additional recurrent mutations, the majority of variants are private, found in single families. The impact of previously reported genotype–phenotype correlations on efficiency of genetic testing was analyzed in our population. Taking our experiment into account, we present our algorithm for genetic testing in CMS. Hum Mutat 33:1474–1484, 2012. © 2012 Wiley Periodicals, Inc.