The island of Chalki, located W of Rhodos, belongs to the East Aegean Islands and is situated at the E part of the South Aegean Island Arc. The flora of Chalki consists of 519 vascular plant taxa, 29 of which are under statutory protection, 22 are Greek endemics and 109 are reported here for the first time. We show that Chalki has the second highest percentage of Greek endemics in the phytogeographical region of the East Aegean Islands. The known distribution of Limonium ocymifolium, L. sitiacum and Phoenix theophrasti is expanded, being reported for the first time for the phytogeographical region of the East Aegean Islands. Finally, we assess for the first time the conservation status of Allium chalkii, a single-island endemic, by biomonitoring its populations.