In a brief letter addressed to Palestinian Authority (PA) President Mahmud Abbas, the signatories condemned the group's declining standing in the Gaza Strip and called for "an end to the internal chaos inside Fatah".
In January, Fatah sustained a heavy blow in the Gaza Strip's first-ever local elections, losing support to rival Hamas, which won 77 of the 118 seats up for grabs.
Fatah won only 26 seats.
The resignations, initiated by Fatah cadres in the Shaikh Radwan neighbourhood of northern Gaza City, appeared to be yet another sign of tension between the old guard and younger elements within the movement.
Last week, 18 party officials in the West Bank submitted their resignation from Fatah, accusing faction bosses of sidelining new talent before legislative elections due in July.
The officials said they felt marginalised by Fatah chiefs who had returned from exile to the occupied territories with late Palestinian leader Yasir Arafat when the Palestinian Authority was set up in 1994.
Old allies of Arafat still dominate key posts at the expense of the new guard who are jostling for party reforms.