Background. Cytoplasmic male sterility (CMS) is a common phenotype in higher plants, which often is associated with rearrangements in mitochondrial DNA (mtDNA), and is widely used to produce hybrid seeds in a variety of valuable crop species. The CMS phenomenon investigations are also promote understanding of a fundamental issue of nuclear-cytoplasmic interactions in the ontogeny of higher plants. In the present study, we analyzed the structural changes in mitochondrial genomes of three alloplasmic lines of sunflower (Helianthus annuus). The investigation was focused on CMS line PET2, as there are very few reports about its mtDNA organization. Methods. The NGS sequencing, de novo assembly, and annotation of sunflower mitochondrial genomes were performed. The comparative analysis of mtDNA of HA89 fertile line and two HA89 CMS lines (PET1, PET2) occurred. Results. The mtDNA of the HA89 fertile line was almost identical to the HA412 line (NC_023337). The comparative analysis of HA89 fertile and CMS (PET1) analog mitochondrial genomes revealed 11852 bp inversion, 4732 bp insertion, 451 bp deletion and 18 variant sites. In mtDNA of HA89 (PET2) CMS line 77 kb translocation, 711 bp and 3780 bp deletions, as well as 1558 bp, 5050 bp, 14330 bp insertions were determined. There are also revealed 83 polymorphic sites sites in the PET2 mitochondrial genome, as compared with the fertile line Discussion. Among the revealed rearrangements the 1558 bp insertion resulted in new open reading frames formation - orf228 and orf246. The orf228 and orf246 could be the main reason for the development of PET2 CMS phenotype, whereas the role of other mtDNA reorganizations in CMS formation is negligible.