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.
Crete, Zakynthos, Mykonos, Lesvos, Paros and Antiparos are among the islands affected.
The announcement comes on the same day the UK government decided not to add Portugal to the list, despite a rise in cases.
Up until now Wales has used the same exemption list as the UK government in England.
The latest Welsh Government rules apply to travellers arriving home in Wales regardless of what part of the UK they returned through – including English airports.
- Greece? Portugal? Spain? What are the quarantine rules?
- Can I still go on holiday to Portugal or Greece?
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.
Greece had not been on the exemption list, except in Scotland, where it was added on Tuesday.
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.
Mr Gething said a report from UK government’s Joint Biosecurity Centre had warned that travel from the countries and islands now listed “constitutes a public health risk”.
The UK government’s Transport Secretary Grant Shapps ruled out any similar changes in England on Thursday.