The reunion, comprising a series of meetings over a week, will be held in a Mount Kumgang resort, near the border.
Thousands of families have been separated with little or no contact since the war ended in 1953.
Reunions have been held sporadically since 1988 and depend on the state of relations between the two countries.
The last reunion was held in February 2014.
This year's meeting comes after an agreement in August that de-escalated tensions sparked by a border explosion that injured South Korean soldiers.
The meetings, organised by the Red Cross, are hugely popular with tens of thousands signing up, but few on each side get chosen and they tend to be elderly.
It is hard to imagine a more bitter-sweet event. The reunion of Korean families who have not seen each other for six decades will be a tumultuous mix of emotions.
There will probably be both tears and laughter as old people divided from those they have not seen for most of their lifetimes get a brief and final meeting.
More than 65,000 South Koreans were eligible for reunions with relatives in the North but only 100 were chosen. Of those hundred, 10 have since died or become too ill to travel.
Read more: The human drama of Korean family reunions
In South Korea participants are picked at random by a computer which takes into account their age and family background.
They also have to sit for interviews and take medical examinations to determine if they are fit to travel.
The first group of about 400 South Koreans, comprising of chosen participants and their accompanying family members, are heading for the first round of meetings running from Tuesday to Thursday, reported Yonhap news agency.
Another 250 will attend the second round of meetings from Saturday to next Monday. Each round comprises of six two-hour sessions.
Many of those attending from South Korea are bringing gifts for their North Korean relatives such as clothes, food, toothpaste, and cash.
Hundreds of South Koreans are travelling to the North for a rare reunion with family members whom they were separated from by the Korean War.