Karlsson crossed the line in a time of 2:07:48.2, clear of her compatriot Linn Svahn who came home 1:48 later, pipping fellow Swede Jonna Sundling on the line by 0.4 seconds to take silver amid a podium sweep for the all-conquering Swedish skiers.
Heidi Weng of Norway narrowly missed out on a medal in front of her home crowd, finishing just over a second behind Sundling in what was an incredibly tight finish for a 50km race. Overall World Cup leader, Jessie Diggins of the USA, finished sixth behind Norway's Karoline Groetting.
Sweden added to the sprint gold Sundling won in Drammen two days earlier, and they dominated the long-distance race despite not relying on Ebba Andersson, who won gold in this event at the Winter Olympics last month.
Andersson was up at the front early on, but suffered a crash around the 20km mark and was cut adrift, eventually finishing 27th, while fellow Swedes Maja Dahlqvist and Moa Ilar were inside the top 20.
After the race, Svahn told Swedish TV of the sweep: "We've talked about doing a triple at a distance at some point. Our coach said he didn't think it would happen in Holmenkollen. It was awesome!"
But the day belonged to Karlsson, who pushed on with 15km to go and distanced the rest of the field for her third World Cup win of the season, adding to the 10km classics she won in Ruka in November and in Lahti earlier in March.
In the men's race, there was no Johannes Hosflot Klaebo, but that didn't stop Norway from not just sweeping the podium, but having the first eight skiers across the line.
Einar Hedegart was the winner in 1:51:38.2, beating Harald Ostberg Amundsen in a sprint finish, ahead of Martin Lowstrom Nyenget.
The final leg of this season's World Cup takes place at Lake Placid, USA, from 20-22 March.