One of China's great historical mysteries has been solved. While it may never be known whether it was the dying Empress Dowager, her loyal eunuch courtier or her commanding army general who was responsible, it has been established that the Guangxu Emperor died of arsenic poisoning in the Water Terrace Pavilion.

Almost a century to the day since the death of China's second-to-last emperor, the discovery will fuel debate over the identity of the murderer who prematurely ended the life of the youthful “Son of Heaven” and with him his ambitions to reform and revive the faltering Qing dynasty.

The Guangxu Emperor was 38 when he died on November 14, 1908 - the day before the ailing Empress Dowager Cixi herself passed away in the nearby Graceful Bird Pavilion of the extended Imperial Palace two weeks shy of her 74th birthday.

The neatness of his death, coming mere hours before that of the woman who had imprisoned him for a decade as punishment for a show of independence, has puzzled historians ever since.

Now, five years of research have established that the Emperor, who had complained of terrible stomach pains, was indeed poisoned. A team that included the administrators of the Western Qing Tombs, the China atomic energy academy and forensic science experts from the Beijing city police took part.

They studied remnants of hair and clothing recovered from his burial chamber, which was robbed in 1936 but is now open to the public. Tests on clothing around his stomach area found that his remains contained as much as 201 milligrammes of arsenic - 2,000 times higher than in a healthy person. A dose of between 60 and 200 milligrammes is considered fatal.

“The amount of arsenic in his body was clearly more than enough to kill him,” the report said. His hair was compared with that of his wife, which contained levels of arsenic 261 times lower.

The researchers ruled out the possibility that he died of chronic poisoning from taking Chinese medicine. Records show that medicines he was prescribed contained traces of arsenic and mercury and could, in theory, have caused his death after being taken over a long period.

“In cases of slow poisonings the roots of the hair of the poisoned person contain smaller amounts of arsenic than the middle section of the hair or the tip, but in the case of Guangxu it was the exact opposite,” the report concluded.

Lei Yi, a prominent Chinese historian, said: “This had been a mystery for 100 years and everyone wanted to crack this puzzle.” But one big question remains unanswered: who killed the Guangxu Emperor?

There are three top suspects, all with strong motives. Top of the list is the Empress Dowager, who chose her three-year-old nephew to become boy-emperor after her own son died. She continued to rule China as regent.

However, the Emperor's attempt to assert himself, modernise China and revive the flagging Qing dynasty by introducing sweeping political change in 1898, alienated conservatives and heralded his political demise.

The Emperor tried to introduce a constitutional monarchy and democracy, to establish a modern university in Beijing, to replace Confucian studies with mathematics and to modernise the military.

The period became known as the “Hundred Days' Reform” - the time it took for the Empress Dowager to organise a coup led by Yuan Shikai, the military commander. Guangxu spent the rest of his life imprisoned in a palace pavilion restoring clocks and watches, learning English and awaiting his chance to return to power. Another possible assassin is Yuan Shikai, who may have feared reprisal for his betrayal of Guangxu after the Empress Dowager died.

The third suspect is Li Lianying, the court's chief eunuch, who learnt that Guangxu had written in his diary of plans for revenge against the Empress Dowager's favourite after her demise - or so legend has it.

The Qing dynasty ended three years after Guangxu's death.

Death and power

— Chongzhen, 33, the last emperor of the Ming dynasty, hanged himself in 1644 as Manchu armies marched in to establish the Qing dynasty – China’s last

— Wu Zetian, the only woman to become empress in her own right is reputed to have killed her baby daughter to throw suspicion on a rival – the chief consort of the 8th-century Tang dynasty emperor

— Taizong, one of China’s greatest emperors, killed two of his brothers and their sons to ensure he would succeed to the throne in the 7th century

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