The North Koreans landed at Incheon International Airport west of Seoul shortly after 9.30am (0030 GMT) on Wednesday and were escorted to unmarked buses waiting inside the airport perimeter.
The buses left in a convoy with curtains drawn to an undisclosed destination through a side gate manned by riot police, witnesses said.
On Tuesday, a first batch of more than 200 North Korean defectors arrived at a military airport south of Seoul under a veil of secrecy.
Both groups arrived on chartered flights from the same unspecified Southeast Asian country, officials said.
The second batch numbered 227, mostly women and children, bringing the total in two days to about 450.
In all, around 450 defectors have
arrived in two days
Officials have described the mass arrivals as "sensitive" and have declined to discuss details of the defections, citing the wishes of the third country for secrecy.
Seoul rejects speculation that it has mounted the low-profile reception to avoid upsetting North Korea in advance of expected inter-Korean cabinet level talks next week.
About 5000 North Koreans have defected to the South since the Korean War ended in 1953. Most have done so in the last three years. More than 1000 have reached South Korea so far this year.
With the highly fortified inter-Korean frontier closed, most cross the poorly guarded northern border into China before fleeing to a third country.
Tens of thousands of North Koreans are said to be in hiding in China and hundreds more are believed to be gathering in various Southeast Asian nations, including Vietnam, Thailand and Cambodia.
"We've so far focussed on helping small numbers of North Korean defectors settle in the country"
unfication minister, South Korea
China refuses to grant North Korean defectors refugee status and considers them illegal economic migrants. If captured, they are repatriated at the request of Pyongyang, where they face severe punishment including internment in camps for political prisoners.
However, Beijing has handled high profile cases differently in the past two years, agreeing to send dozens of North Koreans who managed to enter foreign diplomatic compounds in China, to Seoul via third countries.
Unification Minister Chung Dong-Young on Tuesday said he expected the number of North Koreans in South Korea to double within a few years.
"We've so far focussed on helping small numbers of North Korean defectors settle in the country," Chung said. "We need to review and upgrade the overall policy."