Mitochondrial diseases (MD) are a group of rare inherited disorders, characterized by phenotypic heterogeneity, with hitherto no effective therapeutic options. The aim of this study was to develop a next generation sequencing (NGS) strategy, by using a custom gene panel and whole mitochondrial genome, to identify the disease causing pathogenic variants in 146 patients suspicious of MD. The molecular analysis of this cohort revealed six novel and 15 described pathogenic variants, as well as 26 variants of unknown significance. Our findings are expanding the mutational landscape of these disorders and support the use of a NGS strategy for a higher diagnostic yield. • NGS strategy for mitochondrial diseases diagnosis, with a good balance between cost, time and amount of data for analysis. • Study of genes with a very reduced number of mutations reported. • Identification of novel mutations in nuclear genes associated with mitochondrial diseases. • Evaluation of the first Portuguese patient's cohort suspected of mitochondrial dysfunction investigated by targeted NGS. [ABSTRACT FROM AUTHOR]