Chamomile (Matricaria chamomilla), an important medicinal plant belonging to the Asteraceae family, has a wide distribution in Iran and other parts of the world. The medicinal and pharmacological effects of chamomile are mainly associated with its essential oil content and it is widely used in food, cosmetics and pharmaceutical industries. Despite its wide geographical distribution in Iran, little is known about its molecular genetic diversity and distribution. In this study, intron-exon splice junction (ISJ) markers, including both intron-targeted (IT) and exon-targeted (ET) primers, were used to assess the genetic diversity of thirty-one chamomile populations, including 28 populations from different parts of Iran, one Hungarian population, and two of unknown origin. Twenty-six out of thirty-five primers used in the study, were reliable, producing a total number of 566 sharp and precise bands, of which 557 bands were polymorphic (98%). The average polymorphic information content (PIC) and the average marker index (MI) were calculated at 0.33 and 7.34, respectively. The average total genetic diversity (HT), average genetic diversity within population (HST) and gene differentiation coefficient (Gst) were 0.293, 0.219, and 0.251, respectively. The diversity data revealed that the Matricaria chamomilla species exhibited the closest relationship with the Tripleurospermum disciforme and Tripleurospermum sevanense species.