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Travellers to Wales from mainland Portugal and six Greek islands must self-isolate for 14 days from 04:00 BST Friday.
It is the first time Wales has applied its own quarantine rules.
Gibraltar and French Polynesia will also be removed from the list of countries exempt from the quarantine restrictions.
Mykonos, Zakynthos, Lesvos, Paros, Antiparos and Crete are among the islands affected.
Up until now Wales has used the exemption list as set by the UK government in England.
On Twitter, the UK government's Transport Secretary Grant Shapps ruled out any similar changes in England on Thursday.
Greece had not been on the exemption list, except in Scotland, where it was added on Tuesday.
Travellers to Wales from Zakynthos were asked to self-isolate earlier in the week, but the changes mean those who do not self-isolate face potential fines.
Wales' Health Minister Vaughan Gething said he had taken the decision because of a large number of cases of coronavirus "imported into Wales from tourists returning from the Greek islands".
He said there were more than 20 cases confirmed in passengers on one flight from Zante to Cardiff.
