High resolution melting (HRM) analysis is a powerful detection method for fast, high-throughput post-PCR analysis. A two-step HRM marker system was developed for identification of the N-, S-, R- and T-cytoplasms of onion. In the first step for identification of N-, S-, and R-cytoplasms, one forward primer was designed to the identical sequences of both cox1 and orf725 genes and two reverse primers specific to the polymorphic sequences of cox1 and orf725 genes were used. For the second step breeding lines with N-cytoplasm were evaluated with primers developed from the orfA501 sequence to distinguish between N- and T-cytoplasms. An amplicon with primers to the mitocondrial atp9 gene was used as an internal control. The two-step HRM marker system was tested using 246 onion plants. HRM analysis showed that the most common source of CMS, often used by Russian breeders, is S-cytoplasm, the rarest type of CMS is R-cytoplasm, and the proportion of T-cytoplasm among the analyzed breeding lines was 20.5%.