The Titian, Rest on the Flight into Egypt, was taken on Friday from the state drawing room of the house, seat of the Marquess of Bath. The thieves got away in minutes after using a long ladder to gain access, smashing a first-floor window.
The painting, measuring 18in by 24 (45cm by 60), was on public display and linked to the alarm system, which was triggered at about 9pm. There was no sign of the thieves when police responded within minutes.
Inspector Graham Chivers of Wiltshire Police said they considered it likely that there was a link between the latest raid and that on Lord Pembroke's home in November, when thieves took a portrait of Rembrandt's mother worth £300,000.
The Titian, showing the Virgin Mary cradling the infant Jesus in her arms with Joseph looking on, has been a key attraction at Longleat since it was opened to the public in 1949.
The thieves also made off with a portrait attributed to the circle of the 16th-century artist Joos Van Cleve and a painting by Bonifazio De Pitati. A six-figure reward is being offered for information leading to the return of the Titian, thought to be the only small painting by the artist in private hands.
Lord Bath said security at the house would again be reviewed. Increased security measures were taken recently following the disappearance of a book of Goya engravings worth about £100,000 and the theft of a £2,000 marine chronometer.
Lord Bath said he believed the Titian could have been stolen to order, for private sale or ransom. "It is a particularly fine Titian . It is all very distressing," he said.
The Museums and Galleries Commission reports a sharp increase in robberies from collections open to the public in the past six months.Reuse content