The 1500 participants in the fundraiser started at the Maida Vale Recreation Reserve and travelled along Zig Zag Scenic Drive to Kalamunda, then along the Kalamunda Railway Heritage Trail before tackling the northern most section of the iconic Bibbulmun Track.
After crossing Mundaring Weir, it was on to Mundaring via the Kep Track and on the Railway Reserves Heritage Trail through Darlington and Swan View.
Trekkers will brave the Swan View Tunnel, crossing the National Park Falls, in John Forrest National Park, to join the Eagle View Walk Trail.
Rejoining the Heritage Trail, they will pass through Parkerville, Stoneville and Mt Helena and around Lake Leschenaultia before finishing in Chidlow.
The fastest teams were expected to finish as early as 5pm last night.
Most teams will take about 28-32 hours to finish the 100km, with the majority expected across the line between 7am and 2pm today, while some will finish early tomorrow.
The Perth Hills Discovery Centre will feature as one of the key checkpoints for the South Eastern end of the trail.
The centre yesterday acted as a first-aid point, podiatry post, visitor services and sausage sizzle for support teams, with as many as 2500 trekkers passing through.
Nationally, Oxfam Trailwalker fundraising events are aiming to raise more than $7 million to support vital work to overcome poverty and enable people to bring about positive change in their lives. The Perth event hopes to raise $1.2 million.