Montsho became the first Botswana athlete to win a gold medal at the Commonwealth Games in New Delhi in 2010, but had a forgettable tournament four years later in Glasgow where she tested positive for a banned substance.
With many writing her chances off, Montsho bounced back spectacularly on Wednesday afternoon before a crowd of 35,000 cheering spectators in Gold Coast, Australia.
She appeared to tire, but picked pace on the home stretch to assume the leadership, crossing the line in 50.15.
She beat favorites, Jamaicans, Anastacia Le Roy and Stephenie McPherson into second and third place.
Another Motswana, Christine Botlogetswe fought bravely but could only manage fourth, finishing in 51.17.
This is turning out to be Botswana's most successful Commonwealth Games with three medals thus far, after Isaac Makwala and Baboloki Thebe won gold and silver yesterday.