The Hamilton native won the women's S10 backstroke in a world-record time of one minute 5.90 seconds.
Mortimer also won gold in the 50-metre freestyle event in world-record time last week.
"This feels absolutely amazing," said Mortimer, who also a silver medal in the 200 individual medley earlier at the Games. "I haven't been that fast since I was abled bodied. To be able to beat my times from when I was 14-15 years-old is amazing. I wasn't expecting it but the race went perfectly."
Sophie Pasco of New Zealand, who led Mortimer by a half second at the turn, took the silver in 1:06.69.
"I need to see the clock when I swim backstroke, and I saw that Sophie was ahead of me at the split and I just went for it," Mortimer said.
Shireen Shapiro of South Africa was third in 1:09.02 while Aurelie Rivard of St-Jean-sur-Richelieu, Que., was fifth.
Mortimer ranked third in the preliminaries, but said she was simply following her game plan.
"I went easy in the prelims," she said. "I was trying to strategically place myself a couple of lanes over so I wouldn't be beside the main people and be able to focus on my own race."
The S10 classification is defined as minimal physical impairment. Mortimer broke most of the bones in her feet in a trampoline accident in 2008.
Elsewhere in the pool, Benoit Huot of Montreal won bronze men's S10 backstroke in 1:00.73.
"It wasn't easy but it worked out well for me," said Huot, now with 18 career Paralympic medal through four Games. "This isn't the race I feel the most comfortable in and at the end I wasn't sure I was in the medal contenders. I was really hurting in the last 25 metres."
Justin Zook of the U.S., won the gold medal in a world record 1:00.01. Former world record holder Andre Brasil of Brazil was second in 1:00.11
Earlier in the day, Marco Dispaltro of St-Jerome, Que., and Josh Vander Vies of Vancouver defeated British brothers Stephen and Peter McGuire 8-2 in the bronze-medal boccia match of the BC4 mixed pairs category.
Canada now has 16 medals (four gold, seven silver, five bronze) over seven games of competition. Canada sits 16th in the gold-medal standings and 13th on the overall medal table.
In women's wheelchair basketball, Canada got off to a strong start in its quarter-final against the United States before falling 67-55.
Canada led 21-10 after the first quarter, but the U.S. narrowed that lead to 33-29 by halftime. The Americans continued to outscore Canada over the third and fourth quarters, turning a tight game into a comfortable win.
Jaent McLachlan of North Vancouver, B.C., led Canada with 31 points and 13 rebounds — both game-highs.
In women's goalball, Nancy Morin of Longueuil, Que., scored the game's only goal as Canada beat the United States 1-0. Canada improved to 3-1 and will play Finland in the quarter-finals on Wednesday.
In sailing, Paul Tingley of Halifax finished second for the second race in a row in the single-person keelboat event. Tingley, a gold medallist in Beijing four years ago, is fourth overall with three races to go.
Victoria's John McRoberts and Stacie Louttit were also in fourth after eight races in the two-person keelboat after finishing third in Tuesday's race.
Also Tuesday, it was announced that Canada would host the 2013 International Paralympic Committee swimming championships. The host city has not yet been selected.