DNA markers are integrally connected to the success of molecular breeding and are fundamentally required by breeders to be able to, a. identify new gene sources in the available biodiversity, b. select parents in order to increase heterosis, c. decrease the number of backcross generations for gene introgression breeding programs, and d. carry out marker-assisted selection (MAS). The present research was conducted to examine the efficiency of anchored and non-anchored ISSR molecular markers for grouping 20 bread wheat cultivars introduced in a cold and moderate area of Iran. The results showed an average polymorphism of 82.69% and 75% for anchored and non-anchored ISSR markers, respectively. This result indicates that the anchored ISSR markers have a higher rate of efficiency compared to non-anchored ISSR markers. Therefore, simultaneous grouping of cultivars with the use of anchored and non-anchored ISSR markers can differentiate the cultivars of cold and moderate regions. When anchored and non-anchored ISSR markers were simultaneously applied for cluster analysis and grouping of the individuals, the pattern of genetic diversity aligned with the pattern of geographical distribution, and the cultivars attributed to identical geographical regions were allocated in one group. Based on applied clustering with two anchored and non-anchored markers, it was concluded that Pishtaz and Mahdavi cultivars had less genetic similarity compared to other cultivars.