According to the RSPB two outraged members of the public contacted police after witnessing the alleged incident on the moor in the eastern Cairngorms, ending in the shooting of the hen harrier.
"They explained they were watching for almost three hours as two individuals, armed with shotguns, criss-crossed the moor, with at least one other individual directing them by radio from his vehicle to the location of where the bird was seen perched," the RSPB said.
Gamekeepers' leaders, however, have strongly contested the account of the incident and argue there may be other reasons for the birds' decline.
Hen harriers are supposed to enjoy full legal protection, being the most intensively persecuted of the UK's birds of prey. It is on the International Union for Conservation of Nature's Red List as a species of high conservation concern.
The charity says the incident involving the harrier in the Cairngorms, which happened last year but which has only been made public now, was among a number of examples of illegal persecution of the species in Scotland.
Ian Thomson, RSPB Scotland's Head of Investigations, said: "All the evidence indicates that this appears to have been an appalling, organised killing of one of our rarest birds of prey, which shows a complete disregard of the laws protecting our wildlife. Had it not been for the presence of these two witnesses, no-one would have known about this incident.
"The hen harrier population in Scotland is in trouble, with a 20 per cent decline from 2004-2010. The intolerance shown towards this species on grouse moors, with this latest case being yet another example, gives a clear indication of one of the main causes of this decline."
The RSPB recognises that hen harriers are predators of the red grouse on sporting estates, but say effective and legal techniques, such as diversionary feeding, help.
This involves providing hen harriers with an alternative food source during the period when the adult grouse are feeding their chicks. Over the past seven years, work had been carried out at the Langholm Moor Demonstration Project to reconcile sustainable grouse shooting with maintaining a viable population of hen harriers using the likes of diversionary feeding.
But a spokesman for the Scottish Gamekeepers' Association said: "Our understanding from the case in the Cairngorms is that there is no evidence to support the RSPB's interpretation of events and the RSPB is aware of this.
"In terms of accusing grouse moors alone for the decline of the hen harrier, the RSPB, as a bird charity, could spend donor money more wisely by assessing the bigger picture of harrier decline and the criminal drop in the smaller, less iconic prey birds, rather than spending it on demonising gamekeepers. In Orkney, the hen harrier population declined 70 per cent from 1970, yet there are no gamekeepers or grouse moors.
"Prior to 1995, Langholm Moor was one of the most successful grouse moors in the world but became commercially unviable when a build-up of protected predators, including hen harriers, forced it to close and the gamekeeping staff to be withdrawn."
Tim Baynes, director of the organisation Scottish Land and Estates' moorland group, said sporting estates took their species conservation responsibilities very seriously. "There are other land management techniques employed by estates and gamekeepers, particularly in regard to the control of foxes, which help the hen harrier population," he said.