Centaurea L. is one of the largest genera within the Asteraceae family globally, encompassing approximately 400 to 700 species. This genus presents significant taxonomic challenges. The Iranian flora region comprises 28 sections and 89 species, of which 74 species are native to Iran. Recent studies have introduced several new species. The primary objective of this research was to investigate micro-morphological traits for the identification and differentiation of 16 Centaurea species. Micromorphological studies on plants allow for micro-meter level analysis of leaves, tissues, seeds, etc., using scanning electron microscopy (SEM). In this study, 18 micromorphological traits of the leaf abaxial epidermis, pappus, and achene were examined. The results indicated that among all measured traits, the leaf abaxial epidermis type (ETL), the number of pappus secondary bristles (NFP), abaxial wax density (WDL), pappus width (WOP), and seed surface texture (SST) had the greatest impact on species differentiation. The presence of glandular trichomes (SGL), glandular trichomes diameter (AGL), glandular trichomes density (DGL), achene hairs (ACH), and pappus twist (PAT) were also influential in species discrimination. A principal component analysis (PCA) graph confirmed the effectiveness of these micro-morphological traits in species differentiation.