Few attempts have been made in Iraq to investigate the genetic variation in barley accessions using molecular markers. In this context, the objective of this study was to investigate the diversity of 59 accessions of barley using inter simple sequence repeat (ISSR), conserved DNA-derived polymorphism (CDDP), and start codon targeted (SCoT) markers. A total of 391 amplified polymorphic bands were generated using 45 ISSR, 9 CDDP, and 12 SCoT primers that produced 255, 35, and 101 polymorphic bands, respectively. The average values of gene diversity were 0.77, 0.67, and 0.81 for ISSR, CDDP, and SCoT markers, respectively. The mean values of polymorphism information content for ISSR, CDDP, and SCoT markers were 0.74, 0.63, and 0.80 respectively. The discrimination power of the three approaches for assessing allelic diversity in barley accessions was as follows: SCoT > ISSR > CDDP. The barley accessions were classified and clustered into two main groups. Molecular variance analysis revealed 15, 9, and 14% variability among populations with ISSR, CDDP, and SCoT markers, respectively. The mantel test results revealed that the three molecular marker matrices had significant positive relationships. The SCoT markers have the potential to be useful tools for selecting appropriate parents for breeding program.