The pair, thought to be a mother and a juvenile, have caused much excitement for the team working at RSPB Old Moor.
Heather Bennett, RSPB warden, said: “These images of the playful pair are hard evidence that otters are in the Dearne Valley and look to be very much at home at Old Moor.
“They are a welcome addition to the wildlife and biodiversity in the Dearne Valley that is now even richer in person.”
There has been some previous evidence to suggest otters were living at the nature reserve.
Otter droppings were found there last year, and the team caught a glimpse of the animal, but these images are the first hard evidence.
The landscape of the Dearne Valley was once the most polluted area in northern Europe, but after the pit closures in the 1980s, huge areas are managed for nature and wildlife.
The wetlands were granted SSSI (site of specific scientific interest) in January this year, which makes the area a protected site for wildlife.
Julian Mayston, visitor experience manager at RSPB Old Moor, added: “Otters are extremely sensitive to disturbance and pollution so there presence at Old Moor is testament to the positive changes to the local environment in just over a generation.”