Study of genetic relationships is a prerequisite for plant breeding activities as well as for conservation of genetic resources. In the present study, genetic diversity among and within 15 Iranian native Ajowan(Carum copticum L.) populations were determined using inter simple sequence repeat (ISSR) markers. Twelve selected primers produced 153 discernible bands, with 93 (60.78%) being polymorphic, indicating considerable genetic diversity at the population level. Number of polymorphic bands per primer ranged from 4 to 11 with an average of 7.75. The largest percentage of polymorphic loci was 78.57% for primer UBC818. Based on an un-weighted pair-group method using arithmetic average (UPGMA) clustering algorithm, four distinct groups were established. Results of the principal coordinate analysis (PCoA) corresponded to those obtained through cluster analysis. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly within populations (61%), but variance among populations was only 39%. This study has detected significant genetic differentiation among C. copticum populations. The current study confirmed the importance of molecular studies in detecting genetic variation among genotypes in selecting diverse parents to carry out crossing program successfully.