He becomes Rama X of Thailand's Chakri dynasty, but will not formally be crowned until after his father's cremation
The 64-year-old inherits one of the world's richest monarchies as well as a politically febrile nation, 50 days after King Bhumibol Adulyadej's death.
After weeks of complex palace protocols the prince was invited by the head of the National Legislative Assembly (NLA) to ascend the throne in an event broadcast on all Thai television channels.
"I agree to accept the wishes of the late king... for the benefit of the entire Thai people," said Vajiralongkorn, wearing an official white tunic decorated with medals and a pink sash.
The sombre, ritual-heavy ceremony at his Bangkok palace was attended by the chief of the NLA, junta leader Prayut Chan-O-Cha, and the powerful 96-year-old head of the privy council, Prem Tinsulanonda.
Red-jacketed courtiers looked on as a palace staff member, shuffling on his knees, presented the new king with a microphone through which he delivered his few words of acceptance.
King Vajiralongkorn then prostrated himself, hands pressed together in respect, to a small shrine topped by a picture of his father and mother -- Queen Sirikit Kitiyakara.
He becomes Rama X of Thailand's Chakri dynasty, but will not formally be crowned until after his father's cremation, which is expected next year.
Bhumibol's reign, which ended on October 13, spanned a tumultuous period of Thai history pockmarked by a communist insurgency, coups and street protests.
It also saw breakneck development which has resulted in a huge wealth disparity between a Bangkok-centric elite and the rural poor.
To many Thais, Bhumibol was the only consistent force in a politically combustible country, his image burnished by ritual and shielded by a harsh royal defamation law.
The United States offered its congratulations to the new king, saying it looked forward to strengthening ties with Thailand.
"We offer our best wishes to his majesty and all of the Thai people," the State Department said.
"His father, King Bhumibol, ruled the Kingdom of Thailand with vision and compassion for 70 years and was a great friend of the United States.
"The United States and Thailand enjoy a longstanding, strong, and multifaceted bilateral relationship, and we look forward to deepening that relationship and strengthening the bonds between our two countries and peoples going forward."
Monks chanted blessings at Buddhist temples to mark the new monarch's ascension -- an era-defining moment for most Thais who for seven decades knew only Bhumibol as their king.
Vajiralongkorn does not yet enjoy the same level of popularity.
He spends much of his time outside of the public eye, particularly in southern Germany where he owns property.
He has had three high-profile divorces, while a recent police corruption scandal linked to the family of his previous wife allowed the public a rare glimpse of palace affairs.
Thursday's ascension ends a period of uncertainty since Bhumibol's death prompted by the prince's request to delay his official proclamation so he could mourn with the Thai people.
Thailand's constitutional monarchy has limited formal powers but it draws the loyalty of much of the kingdom's business elite as well as a military that dominates politics through its regular coups.
Analysts say Vajiralongkorn, untested until now, will have to manage competing military cliques.
In a brief televised address after the ceremony, Prime Minister Prayut Chan-O-Cha, who as army chief led the 2014 coup, praised the new king "as the head of the Thai state and heart of the Thai people."
The Thai monarchy is protected from criticism by one of the world's strictest lese majeste laws, carrying up to 15 years in jail for every charge of defaming the king, queen, heir or regent.
Convictions for so-called "112" offences -- named after its criminal code -- have skyrocketed since generals seized power in 2014.
Experts say most have targeted the junta's political opponents, many of whom support the toppled civilian government of Yingluck Shinawatra.
The emergence of Yingluck's brother Thaksin in 2001, a vote-winning billionaire seen by many of the rural poor as their champion, prompted the recent round of political conflict.
The army and royalist establishment have toppled two governments led by the siblings, accusing them of nepotism and corruption.