Staff at the Scottish Sea Life Sanctuary in Oban said the pair were "officially married" at a ceremony in 2011 after becoming inseparable.
They said Isla, who first came to the centre in 2008, died this week aged 14, after suffering stomach problems.
Lewis, who is 11, is now being monitored to ensure he is coping with the loss.
Aquarist Farrah Minchin, who works with the animal car team, said Isla's loss was a "massive blow" for the team.
She said: "Lewis and Isla arrived here at the centre together. Usually otters are quite solitary, they wouldn't normally pair-up for life, but when they arrived they very quickly became inseparable.
"They would groom each other, would look for each other and would sleep together."
Isla was taken to the vet earlier this week after the animal care team noticed she was not eating properly and thought something could be wrong with her teeth or mouth.
However, on closer inspection it was discovered that she had numerous health problems, including peritonitis and a shadow on her lung.
Vets decided not to operate due to her age and the decision was taken to end her life.
Ms Minchin said that since Isla's death on Wednesday, Lewis had been looking for his mate.
She said: "He was at a loss. He was expecting her to come back and kept looking for her.
"He's still swimming on his own side of the pool and not on Isla's, but he does seem to be getting used to things."
Because of his age and an otter's reputation for being aggressive, the animal handling team think it is unlikely Lewis will be paired with another otter.
However, Ms Minchin said that if the right "docile female" came along, Lewis could make a new friend.
Otters can live up to 21 years in captivity, but more generally survive for about nine years in the wild.

The two men were suspected of tampering with the escalator after the incident, report local media.
An escalator at the Langham Place shopping centre suddenly reversed at high speed on Sunday, sending passengers tumbling down.
The reason behind the malfunction is still unknown.
Officials had called in the two technicians, who work for Otis Elevator Company, to assist in investigations hours after the incident.
The escalator had been shut down and all personnel involved in the investigation were ordered not to touch it.
Late on Sunday night however, officials discovered that the escalator's auxiliary braking system had been reactivated, reported local media. Newspaper Apple Daily said the reactivation could have affected the escalator's computer data records.
Officials ordered police to arrest the two men on the spot.
Otis has expressed surprise at the arrests and said it would seek clarification from authorities.
At least 18 people were injured when the escalator, which was going up at Langham Place, suddenly changed direction and started going down at a fast pace.
Videos of the incident show dozens of people losing their balance and tumbling down. Several are seen piled up at the foot of the escalator, with one man lying unconscious on the floor.
Two people are still in hospital and are in a stable condition.
The city government has ordered immediate tests on all escalators which span more than 15m (45 feet) in height.
There has been no official explanation yet, but city officials said a damaged driving chain within the escalator, and a malfunctioning braking device, may have caused the incident.
Escalator engineer Charles Wong told the South China Morning Post that auxiliary brakes should have kicked in if the main brakes had failed, and that it was rare for a driving chain to break.
The mall said that the escalator had last been inspected on Thursday, with no problems detected.
A North American River Otter named Lewis is said to be mourning the death of his "wife" Isla.
[NEXT_CONCEPT]
Two technicians in Hong Kong have been arrested for "perverting the course of justice", after a malfunctioning escalator injured shoppers in a mall.