The onion thrips (Thrips tabaci Lindeman, 1889) is a key pest of a wide range of crops because of its ecological attributes such as polyphagy, high reproduction rate, ability to transmit tospoviruses and resistance to insecticides. Recent studies revealed that T. tabaci is a cryptic species complex and it has three lineages (leek-associated arrhenotokous L1-biotype, leek-associated thelytokous L2-biotype and tobacco-associated arrhenotokous T-biotype), however, the adults remain indistinguishable. T. tabaci individuals were collected from different locations of Hungary to create laboratory colonies from each biotypes. Mitochondrial COI (mtCOI) region was sequenced from morphologically identified individuals. After sequence analysis SNPs were identified and used for CAPS marker development, which were suitable for distinguishing the three T. tabaci lineages. Genetic analysis of the T. tabaci species complex based on mtCOI gene confirmed the three well-known biotypes (L1, L2, T) and a new biotype because the new molecular evidence presented in this study suggests T-biotype of T. tabaci forming two distinct (sub)clades (T1 and T2). This genetic finding indicates that the genetic variability of T. tabaci populations is still not fully mapped. We validated our developed marker on thrips individuals from our thrips colonies. The results demonstrated that the new marker effectively identifies the different T. tabaci biotypes. We believe that our reliable genotyping method will be useful in further studies focusing on T. tabaci biotypes and in pest management by scanning the composition of sympatric T. tabaci populations.