The remains of Nicholas II, his wife and five children, shot by the Bolsheviks in 1918 and hastily buried in the provincial city of Yekaterinburg, are still disputed by the Russian Orthodox Church and some of the family's descendants.
The latest tests, which are still ongoing, could finally lead to the burial of the whole family with full funeral rites.
The influential Church has declared all the slain Romanovs saints -- but has not recognised any of their remains, insisting there are still doubts over their authenticity despite DNA testing by Russian and international scientists.
Russia's government ordered the latest tests on the Church's request as it attempts to finally bury Alexei and Maria.
This time, the Church has been allowed to closely supervise the process.
Tests on the skulls of Nicholas and his wife Alexandra "once more show the authenticity of the 'Yekaterinburg remains,'" the Investigative Committee, which probes major crimes, said in a statement.
"The Church will weigh all the information we have and take its decision in good time," senior Church spokesman Vsevolod Chaplin responded, in comments to Interfax news agency.
The DNA from his skull, which had not previously undergone testing for ethical reasons, also matches that of other bones tested earlier.
Tests on the skull of Nicholas' wife Alexandra matched DNA of descendants of Britain's Queen Victoria, her grandmother.
"Tests will continue," including a comparison with the DNA from bloodied clothing of Tsar Alexander II, Nicholas's grandfather assassinated in 1881, the Investigative Committee said.
The head of the State Archive, Sergei Mironenko, who sits on a government working group on the remains, told Interfax on Wednesday that Alexei and Maria's burial is planned "provisionally for early February (2016)."
Investigators are also preparing to exhume Nicholas II's father, Tsar Alexander III, from the same cathedral.
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