The current research aimed to estimate the evolutionary relationships of ten Centaurea L. species growing naturally in the Duhok City, Kurdistan region of Iraq. The combing Start Codon Targeted (SCoT) markers with Internal Transcribed Spacer (ITS) gene region barcode were performed. To detect the DNA sequence variations and phylogenetic tree reconstruction, the Dice similarity matrix, the unweighted pair group method with arithmetic mean (UPGMA) clustering and Maximum Likelihood (ML) methods were applied. 104 polymorphic bands were scored with an average of 10.4. The Polymorphic Information Content (PIC) and Resolving Power (Rp) values ranged between (0.24 to 0.36) and 3.4 in primer (SCoT1) to 12 in primer (SCoT53) with an average of 0.319 and 5.74 respectively. The lowest similarity value was 0.52 between C. behen L. and C. solstitialis L., while the highest was 0.82 between C. balsamita and C. rigida. The reconstructed polytomous dendrogram was as follows: clade one; C. solstitialis L.; clade 2, C. balsamita Lam. and C. virgata Lam.; clade three subdivide into two subclades: C. iberica Trev. ex Spreng., C. hayalolepis Boiss., C. brugueriana (DC) Hand. Mazz. and C. gigantea Sch. Bip. Ex Boiss., C. regia Boiss., C. rigida Banks & Sol., C. behen Lam. Furthermore, C. brugueriana (DC) Hand. Mazz., C. iberica Trev. ex Spreng, C. behen L., C. solstiotialis L. and C. balsamita Lam. were nested with National Center for Biotechnology Information (NCBI). In contrast, the remaining taxa were mixed with other closely related species. Thus, ScoT markers and ITS DNA barcode were considerably effective for investigating the evolutionary relationships of Centaurea taxa.