The bird, known officially as LF15, and her partner LM12 bred three chicks in both 2015 and 2016 at the Dunkeld reserve.
Lassie arrived back in Scotland on 23 March, six days after her mate.
Viewers from around the world have been watching the pair's progress on the Loch of the Lowes live webcam.
Charlotte Fleming, Perthshire ranger for the Scottish Wildlife Trust, said: "LF15 has settled down to begin incubating, and we would expect the egg to hatch in about six weeks' time.
"It's now clear that the pair's attempts at breeding have been a success and they are on track to repeat the hat-trick of chicks that were hatched in both 2015 and 2016."
A female osprey nicknamed Lassie has laid her first egg of the season at the Scottish Wildlife Trust's Loch of the Lowes nature reserve.