The ANC's OR Tambo chairperson Thandekile Sabisa was re-elected in Mthatha on Sunday, after campaigning as the leader of a faction calling itself the "forces for change", which is informally identified with the Anyone But Zuma (ABZ) campaign within the party.
The group wants party deputy president Kgalema Motlanthe to replace Zuma at the Mangaung elective conference, as part of wholesale changes to senior leadership within the ruling party.
This marks one of the first proper sign of the challenge Zuma faces, as the OR Tambo region is the ANC's second largest regional bloc, after Ethekwini in Kwazulu-Natal – which is still strictly seen as a Zuma stronghold.
The news also comes when senior ANC leaders are calling for renewal in leadership of the party.
Without mentioning Zuma by name, Arts and Culture Minister Paul Mashatile said it was time to reassess the leadership of the ANC.
"No ANC leader is going to lead the organisation for life. It's five years only," it was not a given that you will be re-elected," Mashatile was quoted by the Star as saying at a dinner hosted by the ANC Liliesleaf Farm branch on Saturday.
"Our interest is not an individual. People say Paul is in favour of Tokyo for president, others say I'm in favour of Kgalema for president. We are ANC people," he added.
The ANC in Limpopo, meanwhile, announced a list of "capable leaders" earlier this month August.
While claiming not to be formally nominating any individual, the province mentioned Deputy President Kgalema Motlanthe, Treasurer General Mathews Phosa and new African Union Commission head Nkosazana Dlamini-Zuma.
"There should be nothing that stops us from saying these are the people we think are doing things the best," Limpopo ANC's Makhondo Mathiva told EWN.
Although Limpopo is seen as the province spearheading moves to unseat Zuma, Sabisa's victory and Mashatile's comments show the plan is gaining momentum in the Eastern Cape and Gauteng.
It is also understood that the ABZ campaign has begun receiving support in North Wes, following the Marikana shooting, which saw 34 miners involved in a protracted labour dispute at Lonmin platinum mine gunned down by police on August 16.
Expelled ANC Youth league president Julius Malema – one of Zuma's harshest critics – has been quick to use the tragedy to label Zuma as uncaring and incapable of leading the country as he is out of touch with everyday ANC members.
Besides events at Marikana, Zuma's detractors have found fertile ground for criticism in the president's handling of the Limpopo textbook scandal as well as the "Zumaville" allegations, where the president is reportedly co-ordinating a massive rural development programme just 3.2km from his homestead in Nkandla.
However, Zuma still enjoys majority support in Mpumalanga, the Free State and his home province of KwaZulu-Natal.
For the moment, the ANC is remaining mum on any electioneering currently taking place.
"No matter who says anything, nominations are only open in October and members are only allowed to discuss leadership matters then," ANC spokesperson Keith Khoza told the Mail & Guardian.