It is not known whether Mehsud, a wanted leader of the Pakistan Taliban, is among the dead and injured but he is known to operate in the area.
South Waziristan has become the focus of the Pakistani military's offensive against Taliban sympathisers after it launched an assault on the Swat Valley and Buner in April.
One unnamed security source said Wednesday's second missile strike hit a convoy of vehicles near Janata, a village 50km northwest of the main district town of Wana.
"At least 25 militants have been killed in the US missile strike," he said, adding that the toll is expected to rise.
Wednesday's first air strike using a US drone, which unleashed six missiles on a convoy, struck 35km northeast of Wana.
At least five people were wounded in that raid and officials said its target had been a training camp run by Meshud.
Babar Awan, Pakistan's parliamentary affairs minister, told Al Jazeera that his country has "always objected all the drone attacks" and that was its firm stand.
"Whatever the technology used including the drones, we will not permit anybody else to do this [on Pakistani soil]," he said.
The minister said Pakistan does have cooperation with the US and other friendly countries "in other fields" but not military cooperation.
On Tuesday, a US missile pulverised a compound in South Waziristan, killing up to 16 people.
Wednesday's attacks took to six the number of raids in two weeks against Mehsud and his followers.
Arab leaders of the al-Qaeda network are believed to be also hiding in the region, as well as scores of fighters from nearby countries, especially Afghanistan and Uzbekistan.
The US is thought to have launched more than 40 missiles against targets in the border area since last August, according to a count by The Associated Press news agency.
One attack on the funeral of a dead fighter killed up to 80 people.
Pakistan's army is deploying troops in South Waziristan and launching regular air assaults of its own to try to kill or capture Mehsud, who is blamed for organising many of the suicide attacks in Pakistan over the past few years.