Over the last few decades, molecular markers have been extensively used to study phylogeny, population dynamics, and genome mapping in insects and other taxa. Phylogenetic methods using DNA markers are inexpensive, fast and simple to use, and may help greatly to resolve phylogenetic relationships in groups with problematic taxonomy. However, different markers have various levels of phylogenetic resolution, and it's important to choose the right set of molecular markers for a studied taxonomy level. Acrididae is the most diverse family of grasshoppers. Many attempts to resolve the phylogenetic relationships within it did not result in a clear picture, partially because of the limited number of molecular markers used. We have tested a phylogenetic resolution of three sets of the most commonly utilized mitochondrial molecular markers available for Acrididae sequences in the database: (i) complete protein-coding mitochondrial sequences, (ii) concatenated mitochondrial genes COI, COII, and Cytb, and (iii) concatenated mitochondrial genes COI and COII. We then complemented the analysis by testing the nuclear ITS2 region. Adequate phylogenetic resolution of Acrididae subfamilies can be achieved using three (COI, COII, and Cytb) or more mitochondrial markers. Moreover, we found the ITS2 and concatenated COI/COII markers to be the least informative, providing a poor resolution. All the studied acridids fall into three well-supported phylogenetic groups that include 13 subfamilies. Acridinae, Gomphocerinae, Oedipodinae, and Catantopinae are shown to be polyphyletic, while the remaining subfamilies are in accordance with current Acrididae systematics. Our study provides a basis for more comprehensive phylogenetic analyses of Acrididae on the subfamily and lower levels.