The South Africa women's team crashed out of the 2020 Tokyo Olympic Games qualifiers after they lost 3-2 on penalties to Botswana in Soweto on Tuesday.
On the back of a goalless first-leg draw in Gaborone, the teams played out a barren draw after extra-time before Banyana Banyana lost the ensuing penalty kicks at Orlando Stadium.
In last Friday's first leg, Desiree Ellis' side failed to convert their chances and only managed to secure a goalless draw.
In the return leg, coach Ellis made two changes to the side that started in Botswana, with Leandra Smeda and Busisiwe Ndimeni taking Nothando Vilakazi and Rhoda Mulaudzi's places.
The hosts made an uninspiring start as the visitors almost gained the lead from Andile Dlamini's goalkeeping error in the 22nd minute.
However, Ndimeni brought the home side close to securing the lead but her effort off Lebohang Ramalepe's pass went inches wide 10 minutes later.
Before half time, Jermaine Seoposenwe twice went close from close range for South Africa but her shots could not find the net.
The South Africans were more enterprising in the second half but they failed to find a breakthrough as the visitors held their nerve to force a stalemate at regulation time.
In the extra-time, Banyana continued to ask all the questions but still could not break the resolve of the female Zebras, dragging the contest to a penalty shootout.
In the shootout stage, Banyana missed their final kick, leaving the visitors to claim a 3-2 win.
The second round result means the Cosafa Women's Cup champions will miss the Olympic Games for the first time since 2012, and also for the first time under coach Ellis.
On the other hand, Botswana, who advance at the expense of South Africa, will take on fellow Cosafa rivals Zambia in the third round of the qualifying series later in this month.