In Brazil, there are near 20 genera and almost 120 species of scorpions of which 95% reproduce sexually. Parthenogenetic reproduction, however, may also take place. To gain insight into useful molecular markers in parthenogenetic scorpion species, we studied DNA polymorphism using two molecular approaches: simple sequence repeat anchored polymerase chain reaction (SSR-PCR) and sequencing of the cytochrome C oxidase subunit I of the mitochondrial genome, mtDNA (COXI), of Tityus serrulatus. Three different groups were used: group 1, composed of 1 female and 14 descendants; group 2 with 1 female and 17 descendants, both from the city of Uberlândia, State of Minas Gerais (MG), Brazil, and the third group that consisted of three adult scorpions from the city of Belo Horizonte, MG. The profiles generated by SSR-PCR were identical for all specimens, while partial sequencing of COXI showed the presence of SNPs. After aligning COXI contigs, one of the groups presented 18 SNPs and the second 8 SNPs. The two groups were differentiated by two diagnostic SNPs. We did not find evidence of mitochondrial recombination. The results are in agreement with the parthenogenetic mode of reproduction of this species and sequencing of the COXI gene enabled the separation of scorpions groups.