The Israeli military said Mohammad Odeh was killed in a targeted attack on Tuesday night, just over a week after his predecessor, Izz al-Din al-Haddad, was killed in an Israeli strike on a Gaza apartment block.
The deaths mean there are few militant leaders left in Gaza to lead the group's armed wing, at a time when Israel and Hamas are deadlocked in US-brokered talks to advance President Donald Trump's plan for Gaza's future.
Hours after Odeh's funeral, the Israeli military said it targeted two Hamas militants in a strike in northern Gaza, without disclosing their identity.
At least 10 people were killed, including five children, and 18 wounded in an Israeli strike that targeted a building in Gaza City on Wednesday evening, health officials said.
Sources close to Hamas said Emad Esleem, a local Gaza City commander, was killed in the strike.
The Israeli strike that targeted Odeh also killed his wife and son, his family said. Mourners carried their three bodies, covered in white burial shrouds, past buildings that were bombed out during Israel's two-year military assault on Gaza.
Abu Al-Abd Odeh, one of Odeh's relatives, said Israel's campaign would not stop Palestinians from rising up.
"This journey will not stop and the struggle of the Palestinian people will continue on all levels," he said at a mosque in Gaza City during the funeral.
Gaza health officials say the strike that killed Odeh, his wife and son left at least three others dead and more than 20 wounded. The strike destroyed the upper floor of an apartment building in the Rimal neighbourhood of Gaza City.
Rescue workers dug through the rubble for more possible casualties earlier on Wednesday.
Israeli Prime Minister Benjamin Netanyahu said Odeh headed Hamas' intelligence division at the time of the October 7, 2023 cross-border attack into Israel that triggered the Gaza war.
He was appointed about a week ago to replace Haddad, the group's chief armed commander, who was killed by Israel on May 15.
Sources close to Hamas said Odeh was possibly the last remaining living member of the Hamas armed wing's higher leadership council.
Michael Kobi, of Israel's Institute for National Security Studies, said Hamas had few experienced militant leaders left.
"This is part of the (Israeli) strategy of weakening Hamas, of undermining their cohesion as an organisation," Kobi said.
"When you take down experienced people, then they have a problem to run the organisation effectively."
Israel has killed dozens of Hamas leaders and military officials since the start of the Gaza war and top officials including Netanyahu and his defence minister, Israel Katz, have vowed to kill or capture anyone it says was involved in the October 2023 attacks.
In a statement after Odeh's killing, Katz said Hamas would no longer exercise civilian or military control over Gaza and a plan for what he described as "voluntary migration" from the enclave would also be implemented "at the right time and in the right way".
Palestinians reject any attempt to displace them from Gaza, viewing such comments as reminiscent of the 1948 "Nakba", or catastrophe, when hundreds of thousands of Palestinians were expelled or fled during the war surrounding Israel's creation.
Since the October truce, Israel has killed some 900 Palestinians, according to figures from Gaza health officials that do not distinguish between combatants and civilians.
Four Israeli soldiers have been killed by militants during the same period, the military says.
