Open Doors, a non-denominational Christian group, listed North Korea, Saudi Arabia and Afghanistan as the three toughest countries for Christians last year. They topped the 50-country ranking for 2011 as well.
Syria jumped from 36th to 11th place on the list as its Christian minority, first suspected by rebels of close ties to the Assad government, has increasingly become a target for radical Islamist fighters, the report said.
Ethiopia, which is two-thirds Christian, shot up from 38th to 15th place in the ranking due to a "complex mix of persecution dynamics" including attacks by radical Islamists and reprisals by traditional Christians against new Protestant movements.
Mali came from no listing for 2011 to 7th place because the sharia rule the Islamist Ansar Dine group imposed on the north of the country not only brought harsh punishments for the Muslim majority but also drove the tiny Christian minority, it said.
"There are over 65 countries where Christians are persecuted," said the report released on Tuesday by Open Doors, which began in the 1950s smuggling Bibles into communist states and now works in more than 60 countries.
"An estimated 100 million Christians worldwide are persecuted," the United States-based group said in the report. All but one of the 50 countries in the list - Colombia, which ranked 46th - were in Africa, Asia or the Middle East.
Christianity is the largest and most widely spread faith in the world, with 2.2 billion followers or 32 percent of the world population, according to a report by the Washington-based Pew Forum on Religion and Public Life.
It faces restrictions and hostility in 111 countries around the world, ahead of the 90 countries limiting or harassing the second-largest faith, Islam, another Pew report said.
"In recent years, we've been hearing that Christianity is the most persecuted religion in the world - that sounds right to us," said Open Doors France director Michel Varton at a presentation of the report in Strasbourg.
Leaders of various denominations - including Pope Benedict, whose Roman Catholic followers account for more than half of all Christians - increasingly make this accusation.
It may well be the case given Christianity's size and global spread, but it is hard to produce enough reliable comparative statistics to give it a solid empirical basis.
Some German politicians and human rights groups criticised Chancellor Angela Merkel last November for saying this at a Protestant Church conference there, saying it was pointless to try to rank religions according to how persecuted they were.
Open Doors, which documents cases of persecution of Christians, said its report was based on official studies, news reports and field reports and questionnaires filled out by its staff workers around the world.
Of the top 10 countries on the list - North Korea, Saudi Arabia, Afghanistan, Iraq, Somalia, Maldives, Mali, Iran, Yemen and Eritrea - eight are majority Muslim states threatened by what Open Doors called "Islamic extremism".
North Korea has kept its number one ranking for the past 11 years because it is illegal simply to be a Christian there, it said. Open Doors estimates that up to 70,000 North Koreans have been sent to labour camps for their faith.
The report said second-placed Saudi Arabia, which bans public practice of any faith but Islam, has a growing Christian population because of its migrant workers and some converts it says converted after watching Christian satellite television.
"Christians risk further persecution and oppression in the future due to the rising number of converts and their boldness in sharing their faith," it said.