The Public and Commercial Services union (PCS), said the action would be an "early test" of the government's commitment to champion the Living Wage.
The gallery apologised for its staff's "disappointment" but said it was "in a very difficult financial climate".
PCS General Secretary Mark Serwotka said that many of the National Gallery' staff were "living on poverty pay".
"This will be an early test for the new government to show its commitment to fair pay," he added.
The walk-out is over a 60p pay increase to take staff up to the £7.60 per hour rate called for by campaigners for the London Living Wage.
In February, 100 members of staff walked out over the same row.
A staff pay award introduced by the National gallery in 2009 increased the pay of a number of PCS members among its employees by 3%, but the PCS was not satisfied with the measures.
The union has called on the gallery to reopen negotiations and provide guarantees of future pay increases.
In a statement, the National Gallery said: "In 2010 [it] will continue to do the best that is possible for staff pay within the constraints of affordability and government public sector pay policy.
"The gallery is very sorry that there is disappointment about the award and is particularly supportive of lower paid staff and the need to maintain and improve progression arrangements."
A spokesman for the Department of Culture, Sport and Media said: "Pay levels and industrial relations at our sponsored museums and galleries are entirely a matter for the director and trustees of the museum affected."
Rooms 2-5; 4-5; 14; 16; 17; 19; 28; 30-42; 46 and 51-66 have been closed while the walk-out continues.