The forward settled a drab encounter in the Senegalese capital Dakar when holding his nerve to drill home and finally break Rwanda's resistance.
It is the second time this year the Liverpool forward, heavily linked with a move to Bayern Munich, has converted a spot-kick deep into added time, after his 97th-minute winner against Zimbabwe in Senegal's opening Nations Cup game in Cameroon.
Mane - who also scored the winning penalty in the Nations Cup final - is enjoyable a memorable spell after netting a hat-trick in Saturday's win over Benin to become the Teranga Lions' record goalscorer.
Despite playing in Dakar in what was actually a nominal home tie for Rwanda, the Nations Cup winners - Chelsea goalkeeper Edouard Mendy, Napoli's Kalidou Koulibaly and PSG's Idrissa Gueye among them - struggled for form.
Senegal have played their opening two qualifiers at home, with Rwanda reversing Tuesday's match to enable them to continue renovations on their Amahoro Stadium in Kigali.
The Teranga Lions are five points clear in Group L after just two games, but Benin and Mozambique - who have played just once - meet on Wednesday.
Zambia late show
Earlier on Tuesday, Zambia rebounded from Saturday's opening defeat against Ivory Coast when beating Comoros, who picked up three points off Lesotho on Thursday, 2-1 in Lusaka.
The island nation, who stunned Ghana on their Nations Cup debut earlier this year, looked like they might be set for another upset when taking the lead through El Fardou Ben Nabouhane's early strike.
But the sides were level at the break as Brighton midfielder Enock Mwepu, who missed the end of the Premier League season with injury, converted two minutes into injury time.
Their winner was also late, as Kings Kangwa scored in the 88th-minute winner to secure the 2012 African champions' first points in the group.
Comoros, Zambia and Lesotho are competing for just one spot at the finals, as Ivory Coast are already assured of a place at the tournament as hosts.
The West Africans meet Lesotho in Johannesburg on Thursday.
Stallions continue fine form
Dango Ouattara's competitive Burkina Faso debut came in this year's Africa Cup of Nations, where he impressed
Elsewhere on Tuesday, Dango Ouattara scored twice as Burkina Faso came from behind to beat Eswatini 3-1 and avoid an upset.
Eswatini had grabbed a point against Togo last Friday and took the lead against Burkina Faso through Sabelo Ndzinisa's 65th-minute effort.
Yet their advantage lasted just four minutes as Ouattara equalised before the Lorient forward then made it 2-1 in the 75th minute.
After Eswatini's Lindo Mkhonta was sent off for a second bookable offence, Stephane Aziz Ki rounded off the scoring five minutes before time to extend Burkina Faso's perfect start and keep the Stallions top of Group B.
Cape Verde appeared at their third Nations Cup earlier this year, reaching the Round of 16, and took their first win in the group when beating Togo 2-0 to go second.
Julio Taveres opened the scoring after ten minutes but they had to wait until Jamiro found the net in the fourth minute of added time to secure the win in neutral Morocco.
Togo are winless after two after a disappointing last-gasp draw at home to Eswatini last Friday.
With all teams having played twice, Burkina Faso top the table on six points, followed by Cape Verde (three), Togo and Eswatini (one apiece), with the next two rounds of fixtures scheduled for September.
Upcoming Group H and L matches
Wednesday, 8 June
- Benin v Mozambique (Cotonou, 19:00) - Group L
Thursday, 9 June
- Lesotho v Ivory Coast (Johannesburg, South Africa, 18:00) - Group H
Kick-off times GMT.