The women will begin at 10 a.m. today in Kingston and plan to finish their 305 kilometre swim on Saturday afternoon at Spencer Smith Park in Burlington.
Colleen Shields, 61, Nicole Mallette, 48, Samantha Whiteside, 23, and Rebekah Boscariol, 18, have all completed a solo swim across Lake Ontario.
They each swam the 52-kilometre stretch from Niagara-on-the-Lake to Toronto.
Mona Shariri, 18, the fifth and youngest member of the team by a few months, swam the English Channel last year.
A trip across the length of Lake Ontario by a relay has never been completed.
A key challenge will be dealing with the chilly water.
"With a continous solo swim, you're in the water for the whole time and you never get out," said Whiteside. "So it's not like you're having that hot-to-cold change and that for me is going to be big."
Mallette said the team has been training in the lake since June to get accustomed to Lake Ontario's cold water.
"We started off training in 54 degree [12 C] water. Take your shower that you normally have, put it on cold and multiply that by 10."
The swim will raise money for the charity Because I'm a Girl, which aims to end gender inequality and protect girls' rights.