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Chamomilla recutita (L.) Rausch. is a wide known herbal plant which has many medical attributes and find applications in pharmacy, nutritional and sanitary industries. Estimating genetic diversity in population is very important to protect variety of chamomile species. The objective of this study was characterization of chamomile germplasm using ISSR markers. Among 20 screened ISSR primers, only 5 produced polymorphic and repeatable fragments. In total primers produced 48 fragments out of which 41 (85.4%) were polymorphic. The average PIC value for the amplification products was 0.340. Based on ISSR markers the genetic similarity matrices were produced. The mean genetic similarity was calculated at 0.653. Present study demonstrated that ISSR markers provided a practical and effective method to evaluate the genetic similarity and relationships of chamomile genotypes. Analyzed chamomile genotypes were characterized by quite high genetic similarity; it suggested that there is necessity to find new sources of genetic diversity in chamomile in wild populations.